U.S. patent application number 14/072651 was filed with the patent office on 2014-04-17 for organ-specific proteins and methods of their use.
This patent application is currently assigned to Integrated Diagnostics, Inc.. The applicant listed for this patent is Institute for Systems Biology, Integrated Diagnostics, Inc.. Invention is credited to Patricia M. Beckmann, Leroy Hood, Richard Johnson, Xiaojun Li, Marcello Marelli.
Application Number | 20140106981 14/072651 |
Document ID | / |
Family ID | 39082660 |
Filed Date | 2014-04-17 |
United States Patent
Application |
20140106981 |
Kind Code |
A1 |
Hood; Leroy ; et
al. |
April 17, 2014 |
Organ-Specific Proteins and Methods of Their Use
Abstract
The present invention relates generally to methods for
identifying and using organ-specific proteins and transcripts. The
present invention further provides compositions comprising
organ-specific proteins and transcripts encoding the same,
detection reagents for detecting such proteins and transcripts, and
diagnostic panels, kits and arrays for measuring organ-specific
proteins/transcripts in blood, biological tissue or other
biological fluid.
Inventors: |
Hood; Leroy; (Seattle,
WA) ; Beckmann; Patricia M.; (Hansville, WA) ;
Johnson; Richard; (Mercer Island, WA) ; Marelli;
Marcello; (Seattle, WA) ; Li; Xiaojun;
(Bellevue, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Integrated Diagnostics, Inc.
Institute for Systems Biology |
Seattle
Seattle |
WA
WA |
US
US |
|
|
Assignee: |
Integrated Diagnostics,
Inc.
Seattle
WA
Institute for Systems Biology
Seattle
WA
|
Family ID: |
39082660 |
Appl. No.: |
14/072651 |
Filed: |
November 5, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12376951 |
Jun 30, 2010 |
8586006 |
|
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PCT/US2007/017868 |
Aug 9, 2007 |
|
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14072651 |
|
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60836986 |
Aug 9, 2006 |
|
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Current U.S.
Class: |
506/9 ; 506/12;
506/16; 506/18 |
Current CPC
Class: |
G01N 2800/00 20130101;
A61P 35/00 20180101; G01N 2800/52 20130101; G01N 33/6845 20130101;
G01N 33/68 20130101 |
Class at
Publication: |
506/9 ; 506/18;
506/16; 506/12 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under Grant
Nos. P50 CA097186 and P01 CA085859 awarded by the National Cancer
Institute. The government has certain rights in this invention.
Claims
1. A diagnostic panel comprising: a plurality of detection reagents
wherein each detection reagent is specific for one organ-specific
protein; wherein the organ-specific proteins detected by the
plurality of detection reagents are selected from any one of the
organ-specific protein sets provided in Tables 1-32, 36-45 and
47-79; and wherein the plurality of detection reagents is selected
such that the level of at least one of the organ-specific proteins
detected by the plurality of detection reagents in a blood sample
from a subject afflicted with a disease affecting the organ from
which the organ-specific proteins are derived is above or below a
predetermined normal range.
2. The diagnostic panel of claim 1 wherein the plurality of
detection reagents is selected such that the level of at least two
of the organ-specific proteins detected by the plurality of
detection reagents in a blood sample from a subject afflicted with
a disease affecting the organ from which the organ-specific
proteins are derived is above or below a predetermined normal
range.
3. The diagnostic panel of claim 1 wherein the plurality of
detection reagents is selected such that the level of at least
three of the organ-specific proteins detected by the plurality of
detection reagents in a blood sample from a subject afflicted with
a disease affecting the organ from which the organ-specific
proteins are derived is above or below a predetermined normal
range.
4. The diagnostic panel of claim 1 wherein the plurality of
detection reagents is selected such that the level of at least four
of the organ-specific proteins detected by the plurality of
detection reagents in a blood sample from a subject afflicted with
a disease affecting the organ from which the organ-specific
proteins are derived is above or below a predetermined normal
range.
5. The diagnostic panel of claim 1 wherein the plurality of
detection reagents is between two and 100 detection reagents.
6. The diagnostic panel of claim 1 wherein the organ-specific
proteins detected by the plurality of detection reagents are
selected from any one of the organ-specific protein sets provided
in Tables 1-32, 36-45 and 47-79 and from among the proteins
identified as secreted.
7. The diagnostic panel of claim 1 wherein the organ-specific
proteins detected by the plurality of detection reagents are
selected from any one of the organ-specific protein sets provided
in Tables 1-32, 36-45 and 47-79 and from among the proteins
identified as transmembrane.
8. The diagnostic panel of claim 1 wherein the organ-specific
proteins detected by the plurality of detection reagents are
selected from any one of the organ-specific protein sets provided
in Tables 1-32, 36-45 and 47-79 and from among the proteins with a
specificity of 0.8 or greater.
9. The diagnostic panel of claim 1 wherein the disease affects the
adrenal gland and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 1.
10. The diagnostic panel of claim 1 wherein the disease affects the
bladder and the organ-specific proteins detected by the plurality
of detection reagents are selected from Table 2.
11. The diagnostic panel of claim 1 wherein the disease affects the
bone marrow and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 3.
12. The diagnostic panel of claim 1 wherein the disease affects the
brain amygdala and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 4.
13. The diagnostic panel of claim 1 wherein the disease affects the
colon and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 11.
14. The diagnostic panel of claim 1 wherein the disease affects the
heart and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 12.
15. The diagnostic panel of claim 1 wherein the disease affects the
kidney and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 13.
16. The diagnostic panel of claim 1 wherein the disease affects the
lung and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 14.
17. The diagnostic panel of claim 1 wherein the disease affects the
mammary gland and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 15.
18. The diagnostic panel of claim 1 wherein the disease affects the
peripheral blood and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 16.
19. The diagnostic panel of claim 1 wherein the disease affects the
pancreas and the organ-specific proteins detected by the plurality
of detection reagents are selected from Table 17.
20. The diagnostic panel of claim 1 wherein the disease affects the
peripheral blood and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 18.
21. The diagnostic panel of claim 1 wherein the disease affects the
pituitary gland and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 19.
22. The diagnostic panel of claim 1 wherein the disease affects the
prostate and the organ-specific proteins detected by the plurality
of detection reagents are selected from Table 21.
23. The diagnostic panel of claim 1 wherein the disease affects the
retina and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 22.
24. The diagnostic panel of claim 1 wherein the disease affects the
salivary gland and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 23.
25. The diagnostic panel of claim 1 wherein the disease affects the
Small intestine and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 24.
26. The diagnostic panel of claim 1 wherein the disease affects the
Spinal cord and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 25.
27. The diagnostic panel of claim 1 wherein the disease affects the
spleen and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 26.
28. The diagnostic panel of claim 1 wherein the disease affects the
stomach and the organ-specific proteins detected by the plurality
of detection reagents are selected from Table 27.
29. The diagnostic panel of claim 1 wherein the disease affects the
testis and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 28.
30. The diagnostic panel of claim 1 wherein the disease affects the
thymus and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 29.
31. The diagnostic panel of claim 1 wherein the disease affects the
thyroid and the organ-specific proteins detected by the plurality
of detection reagents are selected from Table 30.
32. The diagnostic panel of claim 1 wherein the disease affects the
uterus and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 32.
33. The diagnostic panel of claim 4 wherein the disease is
Cushing's syndrome.
34. A diagnostic panel comprising: a plurality of detection
reagents wherein each detection reagent is specific for one
organ-specific protein; wherein the organ-specific proteins
detected by the plurality of detection reagents are selected from
two or more of the organ-specific protein sets provided in Tables
1-32, 36-45 and 47-79; and wherein the plurality of detection
reagents is selected such that the level of at least one of the
organ-specific proteins detected by the plurality of detection
reagents in a blood sample from a subject afflicted with a disease
affecting the organs from which the organ-specific proteins are
derived is above or below a predetermined normal range.
35. The diagnostic panel of claim 34 wherein the plurality of
detection reagents is selected such that the level of at least two
of the organ-specific proteins detected by the plurality of
detection reagents in a blood sample from a subject afflicted with
a disease affecting the organs from which the organ-specific
proteins are derived is above or below a predetermined normal
range.
36. The diagnostic panel of claim 34 wherein the plurality of
detection reagents is selected such that the level of at least
three of the organ-specific proteins detected by the plurality of
detection reagents in a blood sample from a subject afflicted with
a disease affecting the organs from which the organ-specific
proteins are derived is above or below a predetermined normal
range.
37. The diagnostic panel of claim 34 wherein the plurality of
detection reagents is selected such that the level of at least four
of the organ-specific proteins detected by the plurality of
detection reagents in a blood sample from a subject afflicted with
a disease affecting the organs from which the organ-specific
proteins are derived is above or below a predetermined normal
range.
38. The diagnostic panel of claim 34 wherein the plurality of
detection reagents is between two and 100 detection reagents.
39. The diagnostic panel of claim 34 wherein the organ-specific
proteins detected by the plurality of detection reagents are
selected from two or more of the organ-specific protein sets
provided in Tables 1-32, 36-45 and 47-79 and from among the
proteins identified as secreted.
40. The diagnostic panel of claim 34 wherein the organ-specific
proteins detected by the plurality of detection reagents are
selected from two or more of the organ-specific protein sets
provided in Tables 1-32, 36-45 and 47-79 and from among the
proteins identified as transmembrane.
41. The diagnostic panel of claim 34 wherein the organ-specific
proteins detected by the plurality of detection reagents are
selected from two or more of the organ-specific protein sets
provided in Tables 1-32, 36-45 and 47-79 and from among the
proteins with a specificity of 0.8 or greater.
42. The diagnostic panel of claim 34 wherein the disease is a
bladder disease and wherein the organ-specific proteins detected by
the plurality of detection reagents are selected from any one or
both of Tables 13 and 2.
43. The diagnostic panel of claim 34 wherein the disease is a
neurological disease and wherein the organ-specific proteins
detected by the plurality of detection reagents are selected from
any one or more of Tables 3, 4, 5, 6, 7, 8 and 9.
44. The diagnostic panel of claim 13 wherein the colon disease is
colon cancer and wherein the organ-specific proteins detected by
the plurality of detection reagents are selected from Table 11.
45. The diagnostic panel of claim 14 wherein the heart disease is
selected from the group consisting of valvular heart disease;
corpulmonale, cardiomyopathy, myocarditis, pericardial disease;
vascular diseases such as atherosclerosis, acute myocardial
infarction, ischemic heart disease and wherein the organ-specific
proteins detected by the plurality of detection reagents are
selected from Table 12.
46. The diagnostic panel of claim 1 wherein the disease affects the
uterus and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 32.
47. The diagnostic panel of claim 1 or claim 34 wherein the
detection reagent comprises an antibody or an antigen-binding
fragment thereof.
48. The diagnostic panel of claim 1 or claim 34 wherein the
detection reagent comprises a DNA or RNA aptamer.
49. The diagnostic panel of claim 1 or claim 34 wherein the
detection reagent comprises an isotope labeled peptide.
50. A method for defining a biological state of a subject
comprising; a. measuring the level of at least two organ-specific
proteins selected from any one of the organ-specific protein sets
provided in Tables 1-32, 36-45 and 47-79 in a blood sample from the
subject; b. comparing the level determined in (a) to a
predetermined normal level of the at least two organ-specific
proteins; wherein a level of at least one of the two organ-specific
proteins that is above or below the predetermined normal level
defines the biological state of the subject.
51. The method of claim 50, wherein the level of the at least two
organ-specific proteins is measured using an immunoassay.
52. The method of claim 51 wherein the immunoassay comprises an
ELISA.
53. The method of claim 50 wherein the level of the at least two
organ-specific proteins is measured using mass spectrometry.
54. The method of claim 50 wherein the level of the at least two
organ-specific proteins is measured using an aptamer capture
assay.
55. A method for defining a biological state of a subject
comprising; a. measuring the level of at least two organ-specific
proteins selected from any two or more of the organ-specific
protein sets provided in Tables 1-32, 36-45 and 47-79 in a blood
sample from the subject; b. comparing the level determined in (a)
to a predetermined normal level of the at least two organ-specific
proteins; wherein a level of at least one of the two organ-specific
proteins that is above or below the predetermined normal level
defines the biological state of the subject.
56. The method of claim 55, wherein the level of the at least two
organ-specific proteins is measured using an immunoassay.
57. The method of claim 56 wherein the immunoassay comprises an
ELISA.
58. The method of claim 55 wherein the level of the at least two
organ-specific proteins is measured using mass spectrometry.
59. The method of claim 55 wherein the level of the at least two
organ-specific proteins is measured using an aptamer capture
assay.
60. A method for defining a disease-associated organ-specific blood
fingerprint comprising; a. measuring the level of at least two
organ-specific proteins selected from any one of the organ-specific
protein sets provided in Tables 1-32, 36-45 and 47-79 in a blood
sample from a subject determined to have a disease affecting the
organ from which the at least two organ-specific proteins are
selected; b. comparing the level of the at least two organ-specific
proteins determined in (a) to a predetermined normal level of the
at least two organ-specific proteins; wherein a level of at least
one of the at least two organ-specific proteins in the blood sample
from the subject determined to have the disease that is below or
above the corresponding predetermined normal level defines the
disease-associated organ-specific blood fingerprint.
61. The method of claim 60 wherein step (a) comprises measuring the
level of at least three organ-specific proteins selected from any
one of the organ-specific protein sets provided in Tables 1-32,
36-45 and 47-79 and wherein a level of at least two of the at least
three organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defines the
disease-associated organ-specific blood fingerprint.
62. The method of claim 60 wherein step (a) comprises measuring the
level of four or more organ-specific proteins selected from any one
of the organ-specific protein sets provided in Tables 1-32, 36-45
and 47-79 and wherein a level of at least three of the four or more
organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defines the
disease-associated organ-specific blood fingerprint.
63. The method of claim 60 wherein step (a) comprises measuring the
level of four or more organ-specific proteins selected from any one
of the organ-specific protein sets provided in Tables 1-32, 36-45
and 47-79 and wherein a level of at least four of the four or more
organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defines the
disease-associated organ-specific blood fingerprint.
64. The method of claim 60 wherein step (a) comprises measuring the
level of five or more organ-specific proteins selected from any one
of the organ-specific protein sets provided in Tables 1-32, 36-45
and 47-79 and wherein a level of at least five of the five or more
organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defines the
disease-associated organ-specific blood fingerprint.
65. The method of claim 60 wherein the level of the at least two
organ-specific proteins is measured using antibodies or
antigen-binding fragments thereof specific for each protein.
66. The method of claim 65 wherein the antibodies or
antigen-binding fragments thereof are monoclonal antibodies.
67. The method of claim 60 wherein the level of the at least two
organ-specific proteins is measured using mass spectrometry.
68. The method of claim 60 wherein the level of the at least two
organ-specific proteins is measured using an aptamer capture
assay.
69. The method of claim 60 wherein the disease is prostate cancer
and the at least two organ-specific proteins are selected from
Table 21.
70. The method of claim 60 wherein the disease is breast cancer and
the at least two organ-specific proteins are selected from Table
15.
71. The method of claim 60 wherein the disease is kidney cancer and
the at least two organ-specific proteins are selected from Table
15.
72. The method of claim 60 wherein the disease is bladder cancer
and the at least two organ-specific proteins are selected from
Table 2.
73. A method for defining a disease-associated organ-specific blood
fingerprint comprising; a. measuring the level of at least two
organ-specific proteins selected from two or more of the
organ-specific protein sets provided in Tables 1-32, 36-45 and
47-79 in a blood sample from a subject determined to have a disease
of interest; b. comparing the level of the at least two
organ-specific proteins determined in (a) to a predetermined normal
level of the at least two organ-specific proteins; wherein a level
of at least one of the at least two organ-specific proteins in the
blood sample from the subject determined to have the disease that
is below or above the corresponding predetermined normal level
defining the disease-associated organ-specific blood
fingerprint.
74. The method of claim 73 wherein step (a) comprises measuring the
level of at least three organ-specific proteins selected from two
or more of the organ-specific protein sets provided in Tables 1-32,
36-45 and 47-79 and wherein a level of at least two of the at least
three organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defining the
disease-associated organ-specific blood fingerprint.
75. The method of claim 73 wherein step (a) comprises measuring the
level of four or more organ-specific proteins selected from two or
more of the organ-specific protein sets provided in Tables 1-32,
36-45 and 47-79 and wherein a level of at least three of the four
or more organ-specific proteins in the blood sample from the
subject determined to have the disease that is below or above the
corresponding predetermined normal level defining the
disease-associated organ-specific blood fingerprint.
76. The method of claim 73 wherein step (a) comprises measuring the
level of four or more organ-specific proteins selected from two or
more of the organ-specific protein sets provided in Tables 1-32,
36-45 and 47-79 and wherein a level of at least four of the four or
more organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defining the
disease-associated organ-specific blood fingerprint.
77. The method of claim 73 wherein step (a) comprises measuring the
level of five or more organ-specific proteins selected from two or
more of the organ-specific protein sets provided in Tables 1-32,
36-45 and 47-79 and wherein a level of at least five of the five or
more organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defining the
disease-associated organ-specific blood fingerprint.
78. A method for detecting perturbation of a normal biological
state in a subject comprising, a) contacting a blood sample from
the subject with a plurality of detection reagents wherein each
detection reagent is specific for one organ-specific protein;
wherein the organ-specific proteins detected by the plurality of
detection reagents are selected from any one of the organ-specific
protein sets provided in Tables 1-32, 36-45 and 47-79; b) measuring
the amount of the organ-specific protein detected in the blood
sample by each detection reagent; and c) comparing the amount of
the organ-specific protein detected in the blood sample by each
detection reagent to a predetermined normal amount for each
respective organ-specific protein; wherein a statistically
significant altered level in one or more of the organ-specific
proteins indicates a perturbation in the normal biological
state.
79. A method for detecting perturbation of a normal biological
state in a subject comprising, a) contacting a blood sample from
the subject with a plurality of detection reagents wherein each
detection reagent is specific for one organ-specific protein;
wherein the organ-specific proteins detected by the plurality of
detection reagents are selected from two or more of the
organ-specific protein sets provided in Tables 1-32, 36-45 and
47-79; b) measuring the amount of the organ-specific protein
detected in the blood sample by each detection reagent; and c)
comparing the amount of the organ-specific protein detected in the
blood sample by each detection reagent to a predetermined normal
amount for each respective organ-specific protein; wherein a
statistically significant altered level in one or more of the
organ-specific proteins indicates a perturbation in the normal
biological state.
80. A method for detecting prostate disease in a subject
comprising, a) contacting a blood sample from the subject with a
plurality of detection reagents wherein each detection reagent is
specific for one prostate-specific protein; wherein the
prostate-specific proteins are selected from the organ-specific
protein set provided in Table 21; b) measuring the amount of the
organ-specific protein detected in the blood sample by each
detection reagent; and c) comparing the amount of the
organ-specific protein detected in the blood sample by each
detection reagent to a predetermined normal control amount for each
respective organ-specific protein; wherein a statistically
significant altered level in one or more of the organ-specific
proteins indicates a perturbation in the normal biological
state.
81. The method of claim 80 wherein the prostate-specific proteins
are selected from those proteins in Table 21 designated as secreted
and with a specificity of 0.9 or greater.
82. The method of claim 80 wherein the prostate disease is selected
from the group consisting of prostate cancer, prostatitis, and
benign prostatic hyperplasia.
83. The method of claim 80 wherein the plurality of detection
reagents comprises at least 2 detection reagents.
84. The method of claim 80 wherein the plurality of detection
reagents comprises at least 3 detection reagents.
85. The method of claim 80 wherein the plurality of detection
reagents comprises at least 4 detection reagents.
86. The method of claim 80 wherein the plurality of detection
reagents comprises at least 5 detection reagents.
87. The method of claim 80 wherein the plurality of detection
reagents comprises at least 6 detection reagents.
88. A method for monitoring a response to a therapy in a subject,
comprising the steps of: (a) measuring in a blood sample obtained
from the subject the level of a plurality of organ-specific
proteins, wherein the plurality of organ-specific proteins are
selected from any one of the organ-specific protein sets provided
in Tables 1-32, 36-45 and 47-79; (b) repeating step (a) using a
blood sample obtained from the subject after undergoing therapy;
and (c) comparing the level of the plurality of organ-specific
proteins detected in step (b) to the amount detected in step (a)
and therefrom monitoring the response to the therapy in the
patient.
89. A method for monitoring a response to a therapy in a subject,
comprising the steps of: (a) measuring in a blood sample obtained
from the subject the level of a plurality of organ-specific
proteins, wherein the plurality of organ-specific proteins are
selected from two or more of the organ-specific protein sets
provided in Tables 1-32, 36-45 and 47-79; (b) repeating step (a)
using a blood sample obtained from the subject after undergoing
therapy; and (c) comparing the level of the plurality of
organ-specific proteins detected in step (b) to the amount detected
in step (a) and therefrom monitoring the response to the therapy in
the patient.
90. A method of imaging an organ, tissue or cells derived from an
organ or tissue, comprising providing an organ-specific probe that
specifically recognizes a sequence of any one or more of the
sequences set forth in Tables 1-32, 36-45 and 47-79, wherein said
probe has attached thereto a label, said label comprising a
detectable marker, administering said probe to an animal and
detecting the location of said probe.
91. An imaging probe comprising an organ-specific probe that
specifically recognizes a sequence of any one or more of the
sequences set forth in Tables 1-32, 36-45 and 47-79, wherein said
probe has attached thereto a label, said label comprising a
detectable marker.
92. A method of targeting an organ, tissue, or cell comprising
providing an organ-specific probe that specifically recognizes a
sequence of any one or more of the sequences set forth in Tables
1-32, 36-45 and 47-79, wherein said probe has attached thereto a
therapeutic agent, said therapeutic agent comprising a radioisotope
or cytotoxic agent.
93. A targeting agent comprising an organ-specific probe that
specifically recognizes a sequence of any one or more of the
sequences set forth in Tables 1-32, 36-45 and 47-79, wherein said
probe has attached thereto a therapeutic agent, said therapeutic
agent comprising a radioisotope or cytotoxic agent.
94. The diagnostic panel of claim 1 wherein the organ-specific
proteins detected by the plurality of detection reagents are
selected from any one of the organ-specific protein sets provided
in Tables 47-79 and from among the proteins identified by MPSS data
and SBS data.
95. The diagnostic panel of claim 34 wherein the organ-specific
proteins detected by the plurality of detection reagents are
selected from two or more of the organ-specific protein sets
provided in Tables 47-79 and from among the proteins identified by
MPSS data and SBS data.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of copending U.S.
application Ser. No. 12/376,951, filed Jun. 30, 2010, which is the
National Stage of International Application No. PCT/US2007/017868,
filed Aug. 9, 2007, which claims benefit of U.S. Provisional
Application No. 60/836,986, filed Aug. 9, 2006. U.S. application
Ser. No. 12/376,951, filed Jun. 30, 2010, International Application
No. PCT/US2007/017868, filed Aug. 9, 2007, and U.S. Provisional
Application No. 60/836,986, filed Aug. 9, 2006, are hereby
incorporated herein by reference in their entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ON COMPACT DISK
[0003] The Sequence Listing associated with this application is
provided on CD-ROM in lieu of a paper copy under AI .sctn.801(a),
and is hereby incorporated by reference into the specification
pursuant to 37 C.F.R. .sctn.1.52(e)(5). Two CD-ROMs are provided
containing identical copies of the sequence listing: CD-ROM No. 3
is labeled "Copy 1 of Sequence Listing," contains the file
655652003300Seqlist.txt which is 135,150,157 bytes and created on
Nov. 3, 2011; and CD-ROM No. 4 is labeled "Copy 2 of Sequence
Listing," contains the file 655652003300Seqlist.txt which is
135,150,157 bytes and created on Nov. 3, 2011.
REFERENCE TO TABLES SUBMITTED ON COMPACT DISK
[0004] Tables 1A-32A, 1B-32B, 36A-45A, 36B-45B, 47A-79A and 47B-79B
associated with this application are provided on CD-ROM in lieu of
a paper copy, and are hereby incorporated by reference into the
specification pursuant to 37 C.F.R. .sctn.1.52(e)(5). Two CD-ROMs
are provided, containing identical copies of the tables, which are
designed to be viewed in landscape presentation: CD-ROM No. 1 is
labeled "Copy 1 of Table," contains the 150 table files which are
20.15 MB combined and created on Aug. 9, 2007; and CD-ROM No. 2 is
labeled "Copy 2 of Tables," contains the 150 table files which are
20.15 MB combined and created on Aug. 9, 2007. Pursuant to 37
C.F.R. .sctn.1.52(e)(5), the names of the files on the CD-ROMs,
their date of creation, and their size in bytes is provided in the
following table.
TABLE-US-00001 Filename Creation Date Size in Bytes Table_1A Aug.
4, 2007 84,515 Table_2A Aug. 4, 2007 59,657 Table_3A Aug. 4, 2007
76,453 Table_4A Aug. 4, 2007 73,910 Table_5A Aug. 4, 2007 146,166
Table_6A Aug. 7, 2007 256,070 Table_7A Aug. 4, 2007 118,077
Table_8A Aug. 4, 2007 488,849 Table_9A Aug. 4, 2007 78,937
Table_10A Aug. 4, 2007 71,119 Table_11A Aug. 4, 2007 70,816
Table_12A Aug. 4, 2007 184,263 Table_13A Aug. 4, 2007 51,918
Table_14A Aug. 4, 2007 171,166 Table_15A Aug. 4, 2007 42,474
Table_16A Aug. 4, 2007 235,766 Table_17A Aug. 4, 2007 12,851
Table_18A Aug. 4, 2007 210,598 Table_19A Aug. 4, 2007 50,457
Table_20A Aug. 4, 2007 133,682 Table_21A Aug. 4, 2007 382,868
Table_22A Aug. 4, 2007 109,625 Table_23A Aug. 4, 2007 57,190
Table_24A Aug. 4, 2007 84,559 Table_25A Aug. 4, 2007 60,631
Table_26A Aug. 4, 2007 292,903 Table_27A Aug. 4, 2007 8,863
Table_28A Aug. 4, 2007 443,581 Table_29A Aug. 4, 2007 120,655
Table_30A Aug. 4, 2007 90,320 Table_31A Aug. 4, 2007 18,462
Table_32A Aug. 4, 2007 26,230 Table_36A Aug. 4, 2007 392,675
Table_37A Aug. 4, 2007 451,715 Table_38A Aug. 4, 2007 65,583
Table_39A Aug. 4, 2007 27,527 Table_40A Aug. 4, 2007 51,618
Table_41A Aug. 4, 2007 21,162 Table_42A Aug. 4, 2007 375,269
Table_43A Aug. 1, 2007 810,314 Table_44A Aug. 1, 2007 127,286
Table_45A Aug. 1, 2007 65,848 Table_47A Aug. 7, 2007 47,132
Table_48A Aug. 7, 2007 456 Table_49A Aug. 7, 2007 9,836 Table_50A
Aug. 7, 2007 453,698 Table_51A Aug. 7, 2007 11,530 Table_52A Aug.
7, 2007 4,298 Table_53A Aug. 7, 2007 102,607 Table_54A Aug. 7, 2007
121,027 Table_55A Aug. 7, 2007 425,448 Table_56A Aug. 7, 2007
130,065 Table_57A Aug. 7, 2007 20,913 Table_58A Aug. 7, 2007 74,589
Table_59A Aug. 7, 2007 54,473 Table_60A Aug. 7, 2007 169,391
Table_61A Aug. 7, 2007 7,348 Table_62A Aug. 7, 2007 145,324
Table_63A Aug. 7, 2007 101,367 Table_64A Aug. 7, 2007 141,026
Table_65A Aug. 7, 2007 160,586 Table_66A Aug. 7, 2007 30,062
Table_67A Aug. 7, 2007 101,479 Table_68A Aug. 7, 2007 844,114
Table_69A Aug. 7, 2007 1,134 Table_70A Aug. 7, 2007 63,850
Table_71A Aug. 7, 2007 9,270 Table_72A Aug. 7, 2007 128,053
Table_73A Aug. 7, 2007 873,964 Table_74A Aug. 7, 2007 14,714
Table_75A Aug. 7, 2007 7,143 Table_76A Aug. 7, 2007 8,950 Table_77A
Aug. 7, 2007 11,550 Table_78A Aug. 7, 2007 286,566 Table_79A Aug.
7, 2007 15,865 Table_1B Aug. 8, 2007 46,350 Table_2B Aug. 4, 2007
28,271 Table_3B Aug. 4, 2007 29,806 Table_4B Aug. 4, 2007 35,795
Table_5B Aug. 4, 2007 57,756 Table_6B Aug. 4, 2007 92,067 Table_7B
Aug. 4, 2007 38,838 Table_8B Aug. 4, 2007 167,183 Table_9B Aug. 4,
2007 34,294 Table_10B Aug. 4, 2007 26,456 Table_11B Aug. 4, 2007
33,269 Table_12B Aug. 4, 2007 79,500 Table_13B Aug. 4, 2007 23,535
Table_14B Aug. 4, 2007 53,563 Table_15B Aug. 4, 2007 20,569
Table_16B Aug. 4, 2007 67,403 Table_17B Aug. 4, 2007 5,666
Table_18B Aug. 4, 2007 58,335 Table_19B Aug. 4, 2007 23,037
Table_20B Aug. 4, 2007 46,979 Table_21B Aug. 4, 2007 115,151
Table_22B Aug. 4, 2007 51,485 Table_23B Aug. 4, 2007 25,090
Table_24B Aug. 4, 2007 31,929 Table_25B Aug. 4, 2007 26,817
Table_26B Aug. 4, 2007 88,101 Table_27B Aug. 4, 2007 4,509
Table_28B Aug. 4, 2007 147,369 Table_29B Aug. 4, 2007 54,981
Table_30B Aug. 4, 2007 27,106 Table_31B Aug. 4, 2007 9,058
Table_32B Aug. 4, 2007 12,498 Table_36B Aug. 4, 2007 118,108
Table_37B Aug. 4, 2007 150,079 Table_38B Aug. 4, 2007 23,135
Table_39B Aug. 4, 2007 13,122 Table_40B Aug. 4, 2007 24,214
Table_41B Aug. 4, 2007 10,752 Table_42B Aug. 4, 2007 112,867
Table_43B Aug. 7, 2007 4,393,320 Table_44B Aug. 7, 2007 634,216
Table_45B Aug. 7, 2007 404,571 Table_47B Aug. 7, 2007 14,056
Table_48B Aug. 7, 2007 333 Table_49B Aug. 7, 2007 3,574 Table_50B
Aug. 7, 2007 157,904 Table_51B Aug. 7, 2007 4,221 Table_52B Aug. 7,
2007 1,953 Table_53B Aug. 7, 2007 38,780 Table_54B Aug. 7, 2007
39,213 Table_55B Aug. 7, 2007 130,256 Table_56B Aug. 7, 2007 26,035
Table_57B Aug. 7, 2007 6,493 Table_58B Aug. 7, 2007 25,070
Table_59B Aug. 7, 2007 17,292 Table_60B Aug. 7, 2007 67,941
Table_61B Aug. 7, 2007 2,816 Table_62B Aug. 7, 2007 49,583
Table_63B Aug. 7, 2007 34,355 Table_64B Aug. 7, 2007 55,195
Table_65B Aug. 7, 2007 57,150 Table_66B Aug. 7, 2007 10,269
Table_67B Aug. 7, 2007 36,082 Table_68B Aug. 7, 2007 283,509
Table_69B Aug. 7, 2007 765 Table_70B Aug. 7, 2007 21,718 Table_71B
Aug. 7, 2007 3,141 Table_72B Aug. 7, 2007 40,961 Table_73B Aug. 7,
2007 293,679 Table_74B Aug. 7, 2007 5,213 Table_75B Aug. 7, 2007
2,837 Table_76B Aug. 7, 2007 3,484 Table_77B Aug. 7, 2007 3,809
Table_78B Aug. 7, 2007 1,015,148 Table_79B Aug. 7, 2007 30,356
BACKGROUND OF THE INVENTION
[0005] 1. Field of the Invention
[0006] The present invention relates generally to organ-specific
proteins and polynucleotides that encode them. In particular the
invention relates to diagnostic and prognostic panels, sets, and
individual agents comprising reagents or probes to detect
organ-specific proteins or polynucleotides and methods of
identifying and using organ-specific proteins.
[0007] 2. Description of the Related Art
[0008] The ability to monitor normal health and to detect the onset
of disease at a very early and treatable stage is critical to
diagnostic medicine. Early detection for most diseases, including
diseases of the lung, cardiovascular disease, cancer, hematological
disease (including most hematological cancers), inflammatory
disorders, metabolic disease and neurological disease may permit
treatment at an earlier stage that will produce healthier and
typically more successful outcomes for the patient. Accordingly,
there is a great need for more sensitive and accurate assays and
methods to measure health and detect disease and monitor treatment
at earlier stages.
[0009] Diagnostic assays are often incapable of identifying truly
informative proteins for analyses and, to be useful, often require
significant changes in protein composition in for example, blood,
at the cellular level to detect the presence of disease or to
define a change in health from normal. Current diagnostic assays
may not detect disease until it has progressed to a stage where it
is too late for effective treatment. For example, most cancers may
be cured if diagnosed at the earliest stage. If cancer is diagnosed
at later or advanced stages, effective treatment may be difficult
or impossible and lead to reduced patient survival. In general,
current diagnostic assays have severe limitations that prevent
early detection and diagnosis.
[0010] In the context of blood protein diagnostics the major
impediment to use in the early detection of disease is that most
proteins are not disease-specific in that multiple organs
synthesize them and different diseases may perturb their expression
in different ways. Moreover, the specific proteins that are
released in the disease state that are markers of the disease may
be difficult to identify or to measure because of the enormous
dynamic range of protein expression in the blood and because of the
enormous protein complexity in the blood. These proteins must be
distinguished from other protein markers found in the blood that
are not likely to be disease markers. Other protein markers that
are present in the blood that are not typically considered
indicators of disease include proteins released due to: cellular
damage, normal cellular turnover, stress responses (liver proteins)
or other slight protein changes in the plasma. Additionally, 22
proteins constitute about 99% of the total blood protein mass.
Indeed, one protein, albumin, comprises about 51% of this total
blood protein mass. Most of these abundant blood proteins are not
useful diagnostic markers. Useful diagnostic proteins are present
in much lower abundance and typically in 1% of the remaining
proteins (Lee et al., Curr Opin in Chem Bio (2006) 10:1-8). Many
proteins are released into the blood following physiological
changes from normal to the disease state and are likely present in
plasma as low abundance proteins. Furthermore, blood proteins
exhibit large differences in the concentration of the most abundant
and least abundant proteins that range over many orders of
magnitude. Proteins are expressed in blood across a range of about
10.sup.10 between the numbers of proteins. This means that one
protein may be present at one copy in a given volume of blood,
whereas another may be present at 10.sup.10 copies (Anderson and
Anderson Mol and Cell Proteomics (2002) 1:845). Low abundance
proteins may be hidden or dwarfed by the more prevalent high
abundance proteins. Additionally, many proteins that are low
abundance proteins are not indicative of disease. Distinguishing
between the low abundance proteins that indicate disease from the
low abundance proteins that are found in the normal cellular state
is a major challenge to modern protein diagnostics. A major
obstacle in diagnostic protein analysis of the blood is the
numerous blood proteins and an inability by current methods to
distinguish proteins from one another. Determining which blood
proteins are predictive of disease at the earliest stages is very
difficult at best, because the diagnostician must distinguish which
low abundance protein is a marker of disease within the mass of
proteins that are circulating in the blood.
[0011] Different approaches for identifying blood proteins are
known in the art and have been used with varying and limited
degrees of success. In particular, two-dimensional gel
electrophoresis (2-DE) has been used for analysis of proteomic
patterns in blood, but it is difficult to resolve large numbers of
proteins such as are expressed in the average cell (up to 10,000
proteins). Moreover, 2-DE is incapable of identifying low abundance
proteins without enrichment techniques. Another method known in the
art for blood protein diagnostics is capillary isoelectric focusing
electrophoresis (CGE) although, the lack of reproducibility of
protein patterns limits its use (Corthals, G. L., et al.
Electrophoresis, (1997), 18:317, Lopez, M. F., and W. F. Patton,
Electrophoresis, (1997), 18:338). Consequently, protein pattern
analysis using techniques such as 2-DE, CGE and other similar
techniques cannot generally be used for the analysis of blood
proteins due to the inability to detect very low abundance
proteins, irreproducible gel patterns, and the inability to
quantify or identify individual spots (e.g., proteins). Further,
the ability to extend these techniques to reproducible, consistent,
easy to use and accurate high throughput diagnostic assays has been
extremely limited. Thus, current assays that detect proteins do not
provide the accuracy to use levels of blood (or other biological
fluid or tissue) proteins, polypeptides or nucleic acids to monitor
health and disease.
[0012] It is evident that a new diagnostic strategy is needed to
distinguish between the many proteins that are found in the blood
that reflect the normal health of a mammal and the organ-specific
proteins that reflect a state of disease.
[0013] For the foregoing reasons, there is a need in the art to
provide diagnostic and prognostic assays, nucleic acid and protein
panels and arrays as well as methods to monitor health and diagnose
disease. The present invention provides compositions, methods and
assays that fulfill these and other needs.
BRIEF SUMMARY OF THE INVENTION
[0014] According to one aspect of the invention, there is provided
a diagnostic panel comprising a plurality of detection reagents
wherein each detection reagent is specific for one organ-specific
protein; wherein the organ-specific proteins detected by the
plurality of detection reagents are selected from any one of the
organ-specific protein sets provided in the Tables herein; and
wherein the plurality of detection reagents is selected such that
the level of at least one of the organ-specific proteins detected
by the plurality of detection reagents in a blood sample from a
subject afflicted with a disease affecting the organ from which the
organ-specific proteins are derived is above or below a
predetermined normal range.
[0015] According to another aspect of the invention, there is
provided a diagnostic panel comprising a plurality of detection
reagents wherein each detection reagent is specific for one
organ-specific protein; wherein the organ-specific proteins
detected by the plurality of detection reagents are selected from
two or more of the organ-specific protein sets provided in the
Tables herein; and wherein the plurality of detection reagents is
selected such that the level of at least one of the organ-specific
proteins detected by the plurality of detection reagents in a blood
sample from a subject afflicted with a disease affecting the organs
from which the organ-specific proteins are derived is above or
below a predetermined normal range.
[0016] According to yet another aspect of the invention, there is
provided a method for defining a biological state of a subject
comprising (a) measuring the level of at least two organ-specific
proteins selected from any one of the organ-specific protein sets
provided in the Tables herein in a blood sample from the subject;
and (b) comparing the level determined in (a) to a predetermined
normal level of the at least two organ-specific proteins; wherein a
level of at least one of the two organ-specific proteins that is
above or below the predetermined normal level defines the
biological state of the subject. In one embodiment of this aspect
of the invention, the level of the at least two organ-specific
proteins is measured using an immunoassay, e.g., by an ELISA assay.
Alternatively, the level of the at least two organ-specific
proteins is measured using mass spectrometry, an aptamer capture
assay or any other suitable technique.
[0017] In another aspect of the invention, there is provided a
method for defining a biological state of a subject comprising (a)
measuring the level of at least two organ-specific proteins
selected from any two or more of the organ-specific protein sets
provided in the Tables herein in a blood sample from the subject;
and (b) comparing the level determined in (a) to a predetermined
normal level of the at least two organ-specific proteins; wherein a
level of at least one of the two organ-specific proteins that is
above or below the predetermined normal level defines the
biological state of the subject. In one embodiment of this aspect
of the invention, the level of the at least two organ-specific
proteins is measured using an immunoassay, e.g., by an ELISA assay.
Alternatively, the level of the at least two organ-specific
proteins is measured using mass spectrometry, an aptamer capture
assay or any other suitable technique.
[0018] In another aspect of the invention, a method is provided for
defining a disease-associated organ-specific blood fingerprint
comprising; (a) measuring the level of at least two organ-specific
proteins selected from any one of the organ-specific protein sets
provided in the Tables herein in a blood sample from a subject
determined to have a disease affecting the organ from which the at
least two organ-specific proteins are selected; and (b) comparing
the level of the at least two organ-specific proteins determined in
(a) to a predetermined normal level of the at least two
organ-specific proteins; wherein a level of at least one of the at
least two organ-specific proteins in the blood sample from the
subject determined to have the disease that is below or above the
corresponding predetermined normal level defines the
disease-associated organ-specific blood fingerprint.
[0019] In one illustrative embodiment of this aspect of the
invention, step (a) comprises measuring the level of at least three
organ-specific proteins selected from any one of the organ-specific
protein sets provided in the Tables herein and wherein a level of
at least two of the at least three organ-specific proteins in the
blood sample from the subject determined to have the disease that
is below or above the corresponding predetermined normal level
defines the disease-associated organ-specific blood
fingerprint.
[0020] In another embodiment, step (a) comprises measuring the
level of four or more organ-specific proteins selected from any one
of the organ-specific protein sets provided in the Tables herein
and wherein a level of at least three of the four or more
organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defines the
disease-associated organ-specific blood fingerprint.
[0021] In yet another embodiment, step (a) comprises measuring the
level of four or more organ-specific proteins selected from any one
of the organ-specific protein sets provided in the Tables herein
and wherein a level of at least four of the four or more
organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defines the
disease-associated organ-specific blood fingerprint.
[0022] In another embodiment, step (a) comprises measuring the
level of five or more organ-specific proteins selected from any one
of the organ-specific protein sets provided in the Tables herein
and wherein a level of at least five of the five or more
organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defines the
disease-associated organ-specific blood fingerprint.
[0023] Another aspect of the invention relates to a method for
defining a disease-associated organ-specific blood fingerprint
comprising; (a) measuring the level of at least two organ-specific
proteins selected from two or more of the organ-specific protein
sets provided in the Tables herein in a blood sample from a subject
determined to have a disease of interest; and (b) comparing the
level of the at least two organ-specific proteins determined in (a)
to a predetermined normal level of the at least two organ-specific
proteins; wherein a level of at least one of the at least two
organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defining the
disease-associated organ-specific blood fingerprint.
[0024] In one embodiment of this aspect of the invention, step (a)
comprises measuring the level of at least three organ-specific
proteins selected from two or more of the organ-specific protein
sets provided in the Tables herein and wherein a level of at least
two of the at least three organ-specific proteins in the blood
sample from the subject determined to have the disease that is
below or above the corresponding predetermined normal level
defining the disease-associated organ-specific blood
fingerprint.
[0025] In another embodiment, step (a) comprises measuring the
level of four or more organ-specific proteins selected from two or
more of the organ-specific protein sets provided in the Tables
herein and wherein a level of at least three of the four or more
organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defining the
disease-associated organ-specific blood fingerprint.
[0026] In another embodiment, step (a) comprises measuring the
level of four or more organ-specific proteins selected from two or
more of the organ-specific protein sets provided in the Tables
herein and wherein a level of at least four of the four or more
organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defining the
disease-associated organ-specific blood fingerprint.
[0027] In yet another embodiment, step (a) comprises measuring the
level of five or more organ-specific proteins selected from two or
more of the organ-specific protein sets provided in the Tables
herein and wherein a level of at least five of the five or more
organ-specific proteins in the blood sample from the subject
determined to have the disease that is below or above the
corresponding predetermined normal level defining the
disease-associated organ-specific blood fingerprint.
[0028] According to another aspect of the invention, there is
provided a method for detecting perturbation of a normal biological
state in a subject comprising, (a) contacting a blood sample from
the subject with a plurality of detection reagents wherein each
detection reagent is specific for one organ-specific protein;
wherein the organ-specific proteins detected by the plurality of
detection reagents are selected from any one of the organ-specific
protein sets provided in the Tables herein; (b) measuring the
amount of the organ-specific protein detected in the blood sample
by each detection reagent; and (c) comparing the amount of the
organ-specific protein detected in the blood sample by each
detection reagent to a predetermined normal amount for each
respective organ-specific protein; wherein a statistically
significant altered level in one or more of the organ-specific
proteins indicates a perturbation in the normal biological
state.
[0029] In another aspect, the invention provides a method for
detecting perturbation of a normal biological state in a subject
comprising, (a) contacting a blood sample from the subject with a
plurality of detection reagents wherein each detection reagent is
specific for one organ-specific protein; wherein the organ-specific
proteins detected by the plurality of detection reagents are
selected from two or more of the organ-specific protein sets
provided in the Tables herein; (b) measuring the amount of the
organ-specific protein detected in the blood sample by each
detection reagent; and (c) comparing the amount of the
organ-specific protein detected in the blood sample by each
detection reagent to a predetermined normal amount for each
respective organ-specific protein; wherein a statistically
significant altered level in one or more of the organ-specific
proteins indicates a perturbation in the normal biological
state.
[0030] Another aspect of the invention provides a method for
detecting prostate disease in a subject comprising, (a) contacting
a blood sample from the subject with a plurality of detection
reagents wherein each detection reagent is specific for one
prostate-specific protein; wherein the prostate-specific proteins
are selected from the organ-specific protein set provided in Table
21; (b) measuring the amount of the organ-specific protein detected
in the blood sample by each detection reagent; and (c) comparing
the amount of the organ-specific protein detected in the blood
sample by each detection reagent to a predetermined normal control
amount for each respective organ-specific protein;
[0031] wherein a statistically significant altered level in one or
more of the organ-specific proteins indicates a perturbation in the
normal biological state.
[0032] In one embodiment of this aspect of the invention, the
prostate-specific proteins are selected from those proteins in
Table 21 designated as secreted and with a specificity of 0.9 or
greater. In another embodiment, the prostate disease is selected
from the group consisting of prostate cancer, prostatitis, and
benign prostatic hyperplasia. In another embodiment, the plurality
of detection reagents comprises at least 2, at least 3, at least 4,
at least 5 or at least 6 detection reagents as described
herein.
[0033] In another aspect of the invention, there is provided a
method for monitoring a response to a therapy in a subject,
comprising the steps of: (a) measuring in a blood sample obtained
from the subject the level of a plurality of organ-specific
proteins, wherein the plurality of organ-specific proteins are
selected from any one of the organ-specific protein sets provided
in the Tables herein; (b) repeating step (a) using a blood sample
obtained from the subject after undergoing therapy; and (c)
comparing the level of the plurality of organ-specific proteins
detected in step (b) to the amount detected in step (a) and
therefrom monitoring the response to the therapy in the
patient.
[0034] In yet another aspect of the invention, there is provide a
method for monitoring a response to a therapy in a subject,
comprising the steps of: (a) measuring in a blood sample obtained
from the subject the level of a plurality of organ-specific
proteins, wherein the plurality of organ-specific proteins are
selected from two or more of the organ-specific protein sets
provided in the Tables herein; (b) repeating step (a) using a blood
sample obtained from the subject after undergoing therapy; and (c)
comparing the level of the plurality of organ-specific proteins
detected in step (b) to the amount detected in step (a) and
therefrom monitoring the response to the therapy in the
patient.
[0035] In the methods of the present invention, the plurality of
detection reagents may be of any suitable or desire number, and
will generally be between about two and 100 detection reagents. In
one embodiment, the plurality of detection reagents is selected
such that the level of at least two, at least three or at least
four of the organ-specific proteins detected by the plurality of
detection reagents in a blood sample from a subject afflicted with
a disease affecting the organ from which the organ-specific
proteins are derived is above or below a predetermined normal
range.
[0036] The organ-specific proteins detected by the plurality of
detection reagents may be selected from any one of the
organ-specific protein sets provided in the Tables herein, and from
among the proteins identified as secreted. In one embodiment, the
organ-specific proteins detected by the plurality of detection
reagents are selected from any one of the organ-specific protein
sets provided in the Tables herein and from among the proteins
identified as transmembrane. In another related embodiment, the
organ-specific proteins detected by the plurality of detection
reagents are selected from any one of the organ-specific protein
sets provided in the Tables herein and from among the proteins with
a specificity of 0.8 or greater. In one embodiment, the
organ-specific proteins detected by the plurality of detection
reagents are selected from any one of the organ-specific protein
sets provided in Tables 47-79 and from among the proteins
identified by MPSS data and SBS data. In this regard, these
proteins are identified in Tables 47-79 by an "&".
[0037] The detection reagent used in the methods of the invention
can be any suitable reagent for detection of the protein or
proteins of interest. For example, in one embodiment, the detection
reagent comprises an antibody (e.g., monoclonal antibody) or an
antigen-binding fragment thereof. In another embodiment, the
detection reagent comprises a DNA or RNA aptamer. In yet another
embodiment, the detection reagent comprises an isotope labeled
peptide.
[0038] The disease or diseases evaluated using the methods
described herein can include essentially any diseases for which the
organ-specific protein sets of the invention provide information of
diagnostic or other medical value.
[0039] For example, in one embodiment, the disease affects the
adrenal gland and the organ-specific proteins detected by the
plurality of detection reagents are selected from Table 1.
[0040] In another embodiment, the disease affects the bladder and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 2. In another embodiment, the
disease is bladder cancer and the at least two organ-specific
proteins are selected from Table 2.
[0041] In another embodiment, the disease affects the bone marrow
and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 3.
[0042] In another embodiment, the disease affects the brain
amygdala and the organ-specific proteins detected by the plurality
of detection reagents are selected from Table 4.
[0043] In another embodiment, the disease affects the colon and the
organ-specific proteins detected by the plurality of detection
reagents are selected from Table 11. In another embodiment, the
colon disease is colon cancer and the organ-specific proteins
detected by the plurality of detection reagents are selected from
Table 11.
[0044] In another embodiment, the disease affects the heart and the
organ-specific proteins detected by the plurality of detection
reagents are selected from Table 12.
[0045] In another embodiment, the disease affects the kidney and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 13. In another embodiment, the
disease is kidney cancer and the at least two organ-specific
proteins are selected from Table 13.
[0046] In another embodiment, the disease affects the lung and the
organ-specific proteins detected by the plurality of detection
reagents are selected from Table 14.
[0047] In another embodiment, the disease affects the mammary gland
and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 15. In another
embodiment, the disease is breast cancer and the at least two
organ-specific proteins are selected from Table 15.
[0048] In another embodiment, the disease affects the peripheral
blood and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 16.
[0049] In another embodiment, the disease affects the pancreas and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 17.
[0050] In another embodiment, the disease affects the peripheral
blood and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 18.
[0051] In another embodiment, the disease affects the pituitary
gland and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 19.
[0052] In another embodiment, the disease affects the prostate and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 21. In another embodiment, the
disease is prostate cancer and the at least two organ-specific
proteins are selected from Table 21.
[0053] In another embodiment, the disease affects the retina and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 22.
[0054] In another embodiment, the disease affects the salivary
gland and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 23.
[0055] In another embodiment, the disease affects the Small
intestine and the organ-specific proteins detected by the plurality
of detection reagents are selected from Table 24.
[0056] In another embodiment, the disease affects the Spinal cord
and the organ-specific proteins detected by the plurality of
detection reagents are selected from Table 25.
[0057] In another embodiment, the disease affects the spleen and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 26.
[0058] In another embodiment, the disease affects the stomach and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 27.
[0059] In another embodiment, the disease affects the testis and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 28.
[0060] In another embodiment, the disease affects the thymus and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 29.
[0061] In another embodiment, the disease affects the thyroid and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 30.
[0062] In another embodiment, the disease affects the uterus and
the organ-specific proteins detected by the plurality of detection
reagents are selected from Table 32.
[0063] In another embodiment, the disease is Cushing's
syndrome.
[0064] In another embodiment, the disease is a bladder disease and
the organ-specific proteins detected by the plurality of detection
reagents are selected from any one or both of Tables 13 and 2.
[0065] In another embodiment, the disease is a neurological disease
and the organ-specific proteins detected by the plurality of
detection reagents are selected from any one or more of Tables 3,
4, 5, 6, 7, 8 and 9.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0066] FIG. 1 is a schematic of the instant invention and provides
a general overview of the steps involved in one embodiment of the
invention.
[0067] FIG. 2 is a photograph of a Western blot of serum from
normal patients, early stage prostate cancer patients and late
stage prostate cancer patients identifying differential expression
of prostate-specific proteins. Serum samples from normal
(1,2,8,13), and from early (3-7) and late (9-12) prostate cancer
patients were resolved by SDS-PAGE and visualized by western
blotting using antibodies directed against STEAP2 and TGM4. The
experiment was conducted as described in Example 4.
BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS
[0068] SEQ ID NOs:1-368 correspond to polynucleotides encoding
adrenal gland-specific proteins as described in Table 1.
[0069] SEQ ID NOs: 369-736 are the amino acid sequences of adrenal
gland-specific proteins as described in Table 1.
[0070] SEQ ID NOs: 737-1028 are the polynucleotide sequences of the
MPSS signature sequences of adrenal gland-specific proteins as
described in Table 1.
[0071] SEQ ID NOs: 1029-1311 correspond to polynucleotides encoding
bladder-specific proteins as described in Table 2.
[0072] SEQ ID NOs: 1312-1594 are the amino acid sequences of
bladder-specific proteins as described in Table 2.
[0073] SEQ ID NOs: 1595-1795 are the polynucleotide sequences of
the MPSS signature sequences of bladder-specific proteins as
described in Table 2.
[0074] SEQ ID NOs: 1796-2094 correspond to polynucleotides encoding
bone marrow-specific proteins as described in Table 3.
[0075] SEQ ID NOs: 2095-2393 are the amino acid sequences of bone
marrow-specific proteins as described in Table 3.
[0076] SEQ ID NOs: 2394-2623 are the polynucleotide sequences of
the MPSS signature sequences of bone marrow-specific proteins as
described in Table 3.
[0077] SEQ ID NOs: 2624-2979 correspond to polynucleotides encoding
brain amygdala-specific proteins as described in Table 4.
[0078] SEQ ID NOs: 2980-3335 are the amino acid sequences of brain
amygdala-specific proteins as described in Table 4.
[0079] SEQ ID NOs: 3336-3579 are the polynucleotide sequences of
the MPSS signature sequences of brain amygdala-specific proteins as
described in Table 4.
[0080] SEQ ID NOs: 3580-4128 correspond to polynucleotides encoding
brain caudate nucleus-specific proteins as described in Table
5.
[0081] SEQ ID NOs: 4129-4677 are the amino acid sequences of brain
caudate nucleus-specific proteins as described in Table 5.
[0082] SEQ ID NOs: 4678-5069 are the polynucleotide sequences of
the MPSS signature sequences of brain caudate nucleus-specific
proteins as described in Table 5.
[0083] SEQ ID NOs: 5070-5903 correspond to polynucleotides encoding
brain cerebellum-specific proteins as described in Table 6.
[0084] SEQ ID NOs: 5904-6737 are the amino acid sequences of brain
cerebellum-specific proteins as described in Table 6.
[0085] SEQ ID NOs: 6738-7211 are the polynucleotide sequences of
the MPSS signature sequences of brain cerebellum-specific proteins
as described in Table 6.
[0086] SEQ ID NOs: 7212-7541 correspond to polynucleotides encoding
brain corpus callosum-specific proteins as described in Table
7.
[0087] SEQ ID NOs: 7542-7871 are the amino acid sequences of brain
corpus callosum-specific proteins as described in Table 7.
[0088] SEQ ID NOs: 7872-8113 are the polynucleotide sequences of
the MPSS signature sequences of brain corpus callosum-specific
proteins as described in Table 7.
[0089] SEQ ID NOs: 8114-9441 correspond to polynucleotides encoding
brain fetal-specific proteins as described in Table 8.
[0090] SEQ ID NOs: 9442-10769 are the amino acid sequences of brain
fetal-specific proteins as described in Table 8.
[0091] SEQ ID NOs: 10770-11903 are the polynucleotide sequences of
the MPSS signature sequences of brain fetal-specific proteins as
described in Table 8.
[0092] SEQ ID NOs: 11904-12159 correspond to polynucleotides
encoding brain hypothalamus-specific proteins as described in Table
9.
[0093] SEQ ID NOs: 12160-12415 are the amino acid sequences of
brain hypothalamus-specific proteins as described in Table 9.
[0094] SEQ ID NOs: 12416-12669 are the polynucleotide sequences of
the MPSS signature sequences of brain hypothalamus-specific
proteins as described in Table 9.
[0095] SEQ ID NOs: 12670-12877 correspond to polynucleotides
encoding brain thalamus-specific proteins as described in Table
10.
[0096] SEQ ID NOs: 12878-13085 are the amino acid sequences of
brain thalamus-specific proteins as described in Table 10.
[0097] SEQ ID NOs: 13086-13264 are the polynucleotide sequences of
the MPSS signature sequences of brain thalamus-specific proteins as
described in Table 10.
[0098] SEQ ID NOs: 13265-13531 correspond to polynucleotides
encoding colon-specific proteins as described in Table 11.
[0099] SEQ ID NOs: 13532-13798 are the amino acid sequences of
colon-specific proteins as described in Table 11.
[0100] SEQ ID NOs: 13799-14035 are the polynucleotide sequences of
the MPSS signature sequences of colon-specific proteins as
described in Table 11.
[0101] SEQ ID NOs: 14036-14449 correspond to polynucleotides
encoding heart-specific proteins as described in Table 12.
[0102] SEQ ID NOs: 14450-14863 are the amino acid sequences of
heart-specific proteins as described in Table 12.
[0103] SEQ ID NOs: 14864-15374 are the polynucleotide sequences of
the MPSS signature sequences of heart-specific proteins as
described in Table 12.
[0104] SEQ ID NOs: 15375-15550 correspond to polynucleotides
encoding kidney-specific proteins as described in Table 13.
[0105] SEQ ID NOs: 15551-15726 are the amino acid sequences of
kidney-specific proteins as described in Table 13.
[0106] SEQ ID NOs: 15727-15904 are the polynucleotide sequences of
the MPSS signature sequences of kidney-specific proteins as
described in Table 13.
[0107] SEQ ID NOs: 15905-16301 correspond to polynucleotides
encoding lung-specific proteins as described in Table 14.
[0108] SEQ ID NOs: 16302-16698 are the amino acid sequences of
lung-specific proteins as described in Table 14.
[0109] SEQ ID NOs: 16669-17022 are the polynucleotide sequences of
the MPSS signature sequences of lung-specific proteins as described
in Table 14.
[0110] SEQ ID NOs: 17023-17182 correspond to polynucleotides
encoding mammary gland-specific proteins as described in Table
15.
[0111] SEQ ID NOs: 17183-17342 are the amino acid sequences of
mammary gland-specific proteins as described in Table 15.
[0112] SEQ ID NOs: 17343-17493 are the polynucleotide sequences of
the MPSS signature sequences of mammary gland-specific proteins as
described in Table 15.
[0113] SEQ ID NOs: 17494-17962 correspond to polynucleotides
encoding monocyte-specific proteins as described in Table 16.
[0114] SEQ ID NOs: 17963-18431 are the amino acid sequences of
monocyte-specific proteins as described in Table 16.
[0115] SEQ ID NOs: 18432-18843 are the polynucleotide sequences of
the MPSS signature sequences of monocyte-specific proteins as
described in Table 16.
[0116] SEQ ID NOs: 18844-18872 correspond to polynucleotides
encoding pancreas-specific proteins as described in Table 17.
[0117] SEQ ID NOs: 18873-18901 are the amino acid sequences of
pancreas-specific proteins as described in Table 17.
[0118] SEQ ID NOs: 18902-18946 are the polynucleotide sequences of
the MPSS signature sequences of pancreas-specific proteins as
described in Table 17.
[0119] SEQ ID NOs: 18947-19350 correspond to polynucleotides
encoding peripheral blood lymphocyte-specific proteins as described
in Table 18.
[0120] SEQ ID NOs: 19351-19754 are the amino acid sequences of
peripheral blood lymphocyte-specific proteins as described in Table
18.
[0121] SEQ ID NOs: 19755-20134 are the polynucleotide sequences of
the MPSS signature sequences of peripheral blood
lymphocyte-specific proteins as described in Table 18.
[0122] SEQ ID NOs: 20135-20275 correspond to polynucleotides
encoding pituitary gland-specific proteins as described in Table
19.
[0123] SEQ ID NOs: 20276-20416 are the amino acid sequences of
pituitary gland-specific proteins as described in Table 19.
[0124] SEQ ID NOs: 20417-20575 are the polynucleotide sequences of
the MPSS signature sequences of pituitary gland-specific proteins
as described in Table 19.
[0125] SEQ ID NOs: 20576-20842 correspond to polynucleotides
encoding placenta-specific proteins as described in Table 20.
[0126] SEQ ID NOs: 20843-21109 are the amino acid sequences of
placenta-specific proteins as described in Table 20.
[0127] SEQ ID NOs: 21110-21435 are the polynucleotide sequences of
the MPSS signature sequences of placenta-specific proteins as
described in Table 20.
[0128] SEQ ID NOs: 21436-22022 correspond to polynucleotides
encoding prostate-specific proteins as described in Table 21.
[0129] SEQ ID NOs: 22023-22609 are the amino acid sequences of
prostate-specific proteins as described in Table 21.
[0130] SEQ ID NOs: 22610-23274 are the polynucleotide sequences of
the MPSS signature sequences of prostate-specific proteins as
described in Table 21.
[0131] SEQ ID NOs: 23275-23605 correspond to polynucleotides
encoding retina-specific proteins as described in Table 22.
[0132] SEQ ID NOs: 23606-23936 are the amino acid sequences of
retina-specific proteins as described in Table 22.
[0133] SEQ ID NOs: 23937-24304 are the polynucleotide sequences of
the MPSS signature sequences of retina-specific proteins as
described in Table 22.
[0134] SEQ ID NOs: 24305-24434 correspond to polynucleotides
encoding salivary gland-specific proteins as described in Table
23.
[0135] SEQ ID NOs: 24435-24564 are the amino acid sequences of
salivary gland-specific proteins as described in Table 23.
[0136] SEQ ID NOs: 24565-24713 are the polynucleotide sequences of
the MPSS signature sequences of salivary gland-specific proteins as
described in Table 23.
[0137] SEQ ID NOs: 24714-24916 correspond to polynucleotides
encoding small intestine-specific proteins as described in Table
24.
[0138] SEQ ID NOs: 24917-2519 are the amino acid sequences of small
intestine-specific proteins as described in Table 24.
[0139] SEQ ID NOs: 25120-25337 are the polynucleotide sequences of
the MPSS signature sequences of small intestine-specific proteins
as described in Table 24.
[0140] SEQ ID NOs: 25338-25477 correspond to polynucleotides
encoding spinal cord-specific proteins as described in Table
25.
[0141] SEQ ID NOs: 25478-25617 are the amino acid sequences of
spinal cord-specific proteins as described in Table 25.
[0142] SEQ ID NOs: 25618-25808 are the polynucleotide sequences of
the MPSS signature sequences of spinal cord-specific proteins as
described in Table 25.
[0143] SEQ ID NOs: 25809-26278 correspond to polynucleotides
encoding spleen-specific proteins as described in Table 26.
[0144] SEQ ID NOs: 26279-26748 are the amino acid sequences of
spleen-specific proteins as described in Table 26.
[0145] SEQ ID NOs: 26749-27329 are the polynucleotide sequences of
the MPSS signature sequences of spleen-specific proteins as
described in Table 26.
[0146] SEQ ID NOs: 27330-27359 correspond to polynucleotides
encoding stomach-specific proteins as described in Table 27.
[0147] SEQ ID NOs: 27360-27389 are the amino acid sequences of
stomach-specific proteins as described in Table 27.
[0148] SEQ ID NOs: 27390-27422 are the polynucleotide sequences of
the MPSS signature sequences of stomach-specific proteins as
described in Table 27.
[0149] SEQ ID NOs: 27423-28424 correspond to polynucleotides
encoding testis-specific proteins as described in Table 28.
[0150] SEQ ID NOs: 28425-29426 are the amino acid sequences of
testis-specific proteins as described in Table 28.
[0151] SEQ ID NOs: 29427-30466 are the polynucleotide sequences of
the MPSS signature sequences of testis-specific proteins as
described in Table 28.
[0152] SEQ ID NOs: 30467-30729 correspond to polynucleotides
encoding thymus-specific proteins as described in Table 29.
[0153] SEQ ID NOs: 30730-30992 are the amino acid sequences of
thymus-specific proteins as described in Table 29.
[0154] SEQ ID NOs: 30993-31348 are the polynucleotide sequences of
the MPSS signature sequences of thymus-specific proteins as
described in Table 29.
[0155] SEQ ID NOs: 31349-31510 correspond to polynucleotides
encoding thyroid-specific proteins as described in Table 30.
[0156] SEQ ID NOs: 31511-31672 are the amino acid sequences of
thyroid-specific proteins as described in Table 30.
[0157] SEQ ID NOs: 31673-31846 are the polynucleotide sequences of
the MPSS signature sequences of thyroid-specific proteins as
described in Table 30.
[0158] SEQ ID NOs: 31847-31888 correspond to polynucleotides
encoding trachea-specific proteins as described in Table 31.
[0159] SEQ ID NOs: 31889-31930 are the amino acid sequences of
trachea-specific proteins as described in Table 31.
[0160] SEQ ID NOs: 31931-32002 are the polynucleotide sequences of
the MPSS signature sequences of trachea-specific proteins as
described in Table 31.
[0161] SEQ ID NOs: 32003-32065 correspond to polynucleotides
encoding uterus-specific proteins as described in Table 32.
[0162] SEQ ID NOs: 32066-32128 are the amino acid sequences of
uterus-specific proteins as described in Table 32.
[0163] SEQ ID NOs: 32129-32206 are the polynucleotide sequences of
the MPSS signature sequences of uterus-specific proteins as
described in Table 32.
[0164] SEQ ID NOs: 25362; 5142; 8639; 15453; 15915; 23547; 23548;
and 17925 correspond to polynucleotides encoding organ-specific
glycosylated proteins identified from a sample of normal human
serum as described in Table 34.
[0165] SEQ ID NOs: 25502; 5976; 9967; 15629; 16312; 23878; 23879;
and 18394 are the amino acid sequences of organ-specific
glycosylated proteins identified from a sample of normal human
serum as described in Table 34.
[0166] The following SEQ ID NOs correspond to the polynucleotides
encoding specific to male organ--prostate-specific proteins as
described in Table 36A identified using MPSS: 21436; 21437; 15907;
21438; 21439; 21440; 21441; 3582; 3583; 3584; 3585; 21442; 13270;
8131; 21443; 1801; 1032; 8135; 14042; 11908; 11909; 11910; 21444;
21445; 21446; 21447; 8144; 8145; 21448; 21449; 21450; 21451; 21452;
21453; 21454; 32207; 21455; 21456; 21457; 21458; 2633; 2634; 2635;
21459; 21460; 21461; 15; 1040; 16; 5101; 21462; 21463; 21464;
21465; 21466; 21467; 21468; 5102; 1814; 1041; 1042; 1043; 21469;
2638; 2639; 1044; 21470; 21471; 21472; 8187; 21473; 21474; 21475;
21476; 21477; 5110; 5111; 3622; 3623; 21478; 2649; 2650; 8198;
15403; 1820; 5122; 5123; 5124; 5125; 8200; 21479; 5126; 1047;
17520; 21480; 21481; 21482; 7238; 29; 21483; 21484; 8226; 21485;
21486; 21487; 21488; 21489; 21490; 32208; 21491; 21492; 21493;
21494; 21495; 21496; 21497; 21498; 21499; 21500; 21501; 18976;
18977; 21502; 21503; 15937; 21504; 1833; 21505; 21506; 21507;
21508; 5145; 21509; 21510; 11951; 21511; 21512; 21513; 15943;
21514; 18983; 18984; 18985; 18986; 18987; 18988; 18989; 18990;
5156; 5157; 21515; 21516; 21517; 8265; 8266; 21518; 21519; 8271;
3670; 21520; 21521; 19008; 21522; 1074; 20603; 19015; 56; 21523;
21524; 21525; 21526; 8300; 21527; 21528; 21529; 21530; 14100;
14101; 21531; 19017; 19018; 32209; 21532; 21533; 21534; 20605;
21535; 21536; 11967; 21537; 14107; 21538; 21539; 8326; 21540; 1869;
1870; 19043; 19044; 19045; 21541; 21542; 21543; 21544; 21545; 7282;
15423; 16001; 21546; 15428; 21547; 21548; 8356; 17589; 21549; 7283;
21550; 21551; 7285; 21552; 21553; 21554; 90; 8366; 8367; 21555;
21556; 21557; 21558; 21559; 21560; 21561; 21562; 21563; 20618;
20619; 21564; 21565; 21566; 21567; 21568; 8408; 5220; 21569; 21570;
21571; 11982; 2699; 21572; 21573; 21574; 1101; 21575; 21576; 1103;
101; 8422; 8423; 21577; 21578; 21579; 21580; 1104; 1885; 21581;
15441; 2705; 21582; 21583; 8458; 14141; 14142; 21584; 21585; 21586;
20622; 20623; 21587; 21588; 21589; 17065; 17066; 17067; 5242; 5243;
8470; 8471; 8476; 8477; 21590; 21591; 21592; 14147; 21593; 1897;
21594; 21595; 21596; 21597; 21598; 21599; 21600; 21601; 21602;
21603; 21604; 7308; 7309; 8508; 3738; 17620; 21605; 21606; 21607;
21608; 21609; 21610; 21611; 21612; 21613; 21614; 21615; 19078;
21616; 21617; 19082; 1121; 121; 122; 123; 124; 5254; 5256; 5257;
5258; 5259; 5260; 21618; 1122; 1123; 21619; 21620; 21621; 8547;
21622; 17636; 17637; 21623; 21624; 8551; 8552; 1132; 21625; 21626;
2730; 2731; 131; 132; 13364; 21627; 21628; 21629; 21630; 21631;
21632; 21633; 21634; 21635; 21636; 21637; 21638; 21639; 7339; 137;
8596; 8597; 8600; 8602; 21640; 1142; 21641; 21642; 21643; 14188;
21644; 21645; 21646; 21647; 5301; 7346; 21648; 21649; 21650; 156;
21651; 1938; 8641; 8642; 8643; 8644; 1939; 21652; 3796; 3797; 3798;
3799; 3800; 3801; 21653; 21654; 8666; 8667; 21655; 2753; 8674;
3821; 2755; 21656; 15459; 5359; 5360; 5361; 5362; 5363; 5364; 5365;
5366; 5367; 5368; 5369; 5370; 5371; 5372; 5373; 5374; 5375; 21657;
21658; 21659; 8700; 14215; 14216; 14217; 14219; 14220; 14222;
21660; 21661; 21662; 2782; 21663; 21664; 21665; 21666; 21667;
21668; 21669; 21670; 21671; 21672; 21673; 21674; 21675; 21676;
21677; 26010; 26011; 26012; 20698; 21678; 21679; 21680; 2792; 5397;
5398; 8735; 8736; 8737; 8738; 8739; 8740; 8741; 8742; 21681; 21682;
21683; 21684; 21685; 21686; 21687; 21688; 21689; 21690; 21691;
21692; 21693; 21694; 32210; 21695; 21696; 21697; 21698; 3867;
21699; 21700; 21701; 21702; 187; 21703; 21704; 21705; 21706; 21707;
21708; 21709; 21710; 21711; 21712; 21713; 21714; 21715; 21716;
32211; 21717; 21718; 21719; 21720; 21721; 21722; 21723; 21724;
21725; 21726; 21727; 21728; 21729; 21730; 21731; 21732; 21733;
21734; 21735; 21736; 21737; 21738; 21739; 21740; 21741; 32212;
21742; 21743; 21744; 21745; 21746; 21747; 21748; 21749; 21750;
21751; 21752; 17790; 19154; 2810; 21753; 21754; 3894; 16099; 16100;
21755; 21756; 21757; 21758; 21759; 21760; 21761; 2821; 1974; 21762;
21763; 21764; 21765; 21766; 21767; 21768; 21769; 21770; 21771;
8866; 2823; 2824; 2825; 2826; 2827; 2828; 2829; 2830; 21772; 21773;
21774; 2831; 21775; 2832; 8889; 8890; 16108; 5468; 21776; 21777;
8896; 21778; 21779; 21780; 21781; 21782; 21783; 21784; 211; 21785;
20238; 14271; 21786; 21787; 21788; 21789; 21790; 32213; 17117;
21791; 21792; 21793; 21794; 21795; 21796; 21797; 15493; 21798;
21799; 21800; 21801; 21802; 21803; 5477; 3915; 21804; 21805; 21806;
21807; 21808; 21809; 5479; 5480; 5481; 5482; 5483; 5484; 5485;
5486; 5487; 5488; 5489; 5490; 5491; 5492; 5493; 5494; 5495; 5496;
5497; 5498; 5499; 5500; 5501; 5502; 5503; 5504; 5505; 5506; 5507;
5508; 5509; 5510; 5511; 5512; 5513; 5514; 5515; 5516; 5517; 5518;
5519; 5520; 5521; 5522; 5523; 5524; 5525; 5526; 5527; 5528; 5529;
5530; 5531; 5532; 5533; 5534; 5535; 5536; 5537; 5538; 5539; 5540;
5541; 5542; 5543; 5544; 5545; 5546; 5547; 5548; 5549; 5550; 5551;
5552; 5553; 5554; 5555; 5556; 5557; 5558; 5559; 5560; 5561; 5562;
5563; 5564; 5565; 5566; 5567; 5568; 5569; 5570; 5571; 5572; 5573;
5574; 5575; 5576; 5577; 5578; 5579; 5580; 5581; 5582; 5583; 5584;
5585; 5586; 5587; 5588; 5589; 5590; 5591; 5592; 5593; 5594; 5595;
5596; 5597; 5598; 5599; 5600; 5601; 5602; 5603; 5604; 5605; 5606;
5607; 5608; 5609; 5610; 5611; 5612; 5613; 5614; 5615; 16124; 8916;
8917; 21810; 21811; 21812; 21813; 21814; 21815; 25424; 21816;
16133; 21817; 21818; 21819; 21820; 17820; 17821; 14293; 21821;
21822; 21823; 21824; 20745; 20746; 20747; 17123; 21825; 21826;
8959; 2859; 21827; 21828; 21829; 21830; 21831; 21832; 21833; 21834;
21835; 3948; 21836; 21837; 21838; 1201; 1996; 16141; 21839; 21840;
21841; 21842; 17129; 17130; 3952; 21843; 21844; 12815; 32214;
21845; 21846; 32215; 21847; 21848; 21849; 2873; 2874; 2875; 21850;
3964; 21851; 21852; 5682; 5683; 5684; 21853; 21854; 21855; 21856;
21857; 21858; 21859; 3972; 3973; 21860; 21861; 21862; 21863; 5686;
7436; 21864; 21865; 21866; 21867; 21868; 21869; 2881; 2882; 9061;
9062; 9063; 21870; 21871; 21872; 265; 266; 21873; 21874; 14343;
14344; 14345; 3986; 21875; 21876; 21877; 21878; 21879; 21880;
21881; 2891; 16168; 19218; 19219; 21882; 21883; 19220; 19221;
19222; 19223; 21884; 21885; 19224; 19225; 19226; 19227; 21886;
7452; 7453; 7454; 7455; 9097; 21887; 21888; 21889; 16170; 16171;
21890; 21891; 21892; 21893; 21894; 32216; 21895; 21896; 9119;
21897; 21898; 21899; 21900; 21901; 21902; 21903; 9139; 21904; 5739;
21905; 21906; 17888; 19241; 19243; 19244; 19245; 4016; 9156; 9157;
4017; 4018; 7468; 21907; 21908; 4021; 19256; 21909; 21910; 16178;
9166; 9167; 14363; 2039; 19261; 5753; 9179; 9180; 9181; 9182; 9183;
21911; 16188; 21912; 21913; 21914; 21915; 21916; 21917; 9187; 9220;
21918; 21919; 21920; 21921; 14372; 2054; 21922; 21923; 21924;
21925; 19280; 21926; 21927; 4035; 1258; 2911; 2912; 32217; 5777;
21928; 7483; 7484; 7485; 21929; 21930; 1262; 1263; 1264; 1265;
1266; 1267; 9264; 9265; 9266; 9267; 9268; 9269; 9270; 13490; 21931;
21932; 25454; 32218; 21933; 2922; 4051; 4052; 21934; 21935; 21936;
21937; 21938; 21939; 21940; 21941; 1270; 21942; 5789; 4061; 21943;
19299; 1277; 21944; 21945; 21946; 21947; 9308; 20813; 21948; 327;
21949; 21950; 21951; 21952; 21953; 21954; 21955; 21956; 21957;
21958; 21959; 4075; 21960; 16229; 32219; 21961; 21962; 21963;
21964; 21965; 336; 21966; 21967; 21968; 21969; 21970; 21971; 21972;
9326; 14410; 21973; 9329; 21974; 21975; 9332; 21976; 21977; 21978;
21979; 12857; 21980; 21981; 21982; 21983; 13525; 343; 21984; 21985;
20825; 16246; 21986; 15540; 15541; 7521; 21987; 2081; 21988; 12863;
21989; 21990; 21991; 13527; 13528; 21992; 16257; 20830; 21993;
21994; 21995; 19328; 19329; 19330; 21996; 20833; 21997; 21998;
21999; 22000; 32220; 22001; 17955; 17956; 17957; 22002; 22003;
22004; 22005; 22006; 361; 12869; 12870; 22007; 17178; 16290; 22008;
22009; 22010; 22011; 22012; 9416; 9417; 22013; 22014; 22015; 22016;
22017; 22018; 22019; 22020; 22021; 22022.
[0167] The following SEQ ID NOs correspond to the amino acid
sequences of specific to male organ--prostate-specific proteins as
described in Table 36A identified using MPSS: 22023; 22024; 16304;
22025; 22026; 22027; 22028; 4131; 4132; 4133; 4134; 22029; 13537;
9459; 22030; 2100; 1315; 9463; 14456; 12164; 12165; 12166; 22031;
22032; 22033; 22034; 9472; 9473; 22035; 22036; 22037; 22038; 22039;
22040; 22041; 32221; 22042; 22043; 22044; 22045; 2989; 2990; 2991;
22046; 22047; 22048; 383; 1323; 384; 5935; 22049; 22050; 22051;
22052; 22053; 22054; 22055; 5936; 2113; 1324; 1325; 1326; 22056;
2994; 2995; 1327; 22057; 22058; 22059; 9515; 22060; 22061; 22062;
22063; 22064; 5944; 5945; 4171; 4172; 22065; 3005; 3006; 9526;
15579; 2119; 5956; 5957; 5958; 5959; 9528; 22066; 5960; 1330;
17989; 22067; 22068; 22069; 7568; 397; 22070; 22071; 9554; 22072;
22073; 22074; 22075; 22076; 22077; 32222; 22078; 22079; 22080;
22081; 22082; 22083; 22084; 22085; 22086; 22087; 22088; 19380;
19381; 22089; 22090; 16334; 22091; 2132; 22092; 22093; 22094;
22095; 5979; 22096; 22097; 12207; 22098; 22099; 22100; 16340;
22101; 19387; 19388; 19389; 19390; 19391; 19392; 19393; 19394;
5990; 5991; 22102; 22103; 22104; 9593; 9594; 22105; 22106; 9599;
4219; 22107; 22108; 19412; 22109; 1357; 20870; 19419; 424; 22110;
22111; 22112; 22113; 9628; 22114; 22115; 22116; 22117; 14514;
14515; 22118; 19421; 19422; 32223; 22119; 22120; 22121; 20872;
22122; 22123; 12223; 22124; 14521; 22125; 22126; 9654; 22127; 2168;
2169; 19447; 19448; 19449; 22128; 22129; 22130; 22131; 22132; 7612;
15599; 16398; 22133; 15604; 22134; 22135; 9684; 18058; 22136; 7613;
22137; 22138; 7615; 22139; 22140; 22141; 458; 9694; 9695; 22142;
22143; 22144; 22145; 22146; 22147; 22148; 22149; 22150; 20885;
20886; 22151; 22152; 22153; 22154; 22155; 9736; 6054; 22156; 22157;
22158; 12238; 3055; 22159; 22160; 22161; 1384; 22162; 22163; 1386;
469; 9750; 9751; 22164; 22165; 22166; 22167; 1387; 2184; 22168;
15617; 3061; 22169; 22170; 9786; 14555; 14556; 22171; 22172; 22173;
20889; 20890; 22174; 22175; 22176; 17225; 17226; 17227; 6076; 6077;
9798; 9799; 9804; 9805; 22177; 22178; 22179; 14561; 22180; 2196;
22181; 22182; 22183; 22184; 22185; 22186; 22187; 22188; 22189;
22190; 22191; 7638; 7639; 9836; 4287; 18089; 22192; 22193; 22194;
22195; 22196; 22197; 22198; 22199; 22200; 22201; 22202; 19482;
22203; 22204; 19486; 1404; 489; 490; 491; 492; 6088; 6090; 6091;
6092; 6093; 6094; 22205; 1405; 1406; 22206; 22207; 22208; 9875;
22209; 18105; 18106; 22210; 22211; 9879; 9880; 1415; 22212; 22213;
3086; 3087; 499; 500; 13631; 22214; 22215; 22216; 22217; 22218;
22219; 22220; 22221; 22222; 22223; 22224; 22225; 22226; 7669; 505;
9924; 9925; 9928; 9930; 22227; 1425; 22228; 22229; 22230; 14602;
22231; 22232; 22233; 22234; 6135; 7676; 22235; 22236; 22237; 524;
22238; 2237; 9969; 9970; 9971; 9972; 2238; 22239; 4345; 4346; 4347;
4348; 4349; 4350; 22240; 22241; 9994; 9995; 22242; 3109; 10002;
4370; 3111; 22243; 15635; 6193; 6194; 6195; 6196; 6197; 6198; 6199;
6200; 6201; 6202; 6203; 6204; 6205; 6206; 6207; 6208; 6209; 22244;
22245; 22246; 10028; 14629; 14630; 14631; 14633; 14634; 14636;
22247; 22248; 22249; 3138; 22250; 22251; 22252; 22253; 22254;
22255; 22256; 22257; 22258; 22259; 22260; 22261; 22262; 22263;
22264; 26480; 26481; 26482; 20965; 22265; 22266; 22267; 3148; 6231;
6232; 10063; 10064; 10065; 10066; 10067; 10068; 10069; 10070;
22268; 22269; 22270; 22271; 22272; 22273; 22274; 22275; 22276;
22277; 22278; 22279; 22280; 22281; 32224; 22282; 22283; 22284;
22285; 4416; 22286; 22287; 22288; 22289; 555; 22290; 22291; 22292;
22293; 22294; 22295; 22296; 22297; 22298; 22299; 22300; 22301;
22302; 22303; 32225; 22304; 22305; 22306; 22307; 22308; 22309;
22310; 22311; 22312; 22313; 22314; 22315; 22316; 22317; 22318;
22319; 22320; 22321; 22322; 22323; 22324; 22325; 22326; 22327;
22328; 32226; 22329; 22330; 22331; 22332; 22333; 22334; 22335;
22336; 22337; 22338; 22339; 18259; 19558; 3166; 22340; 22341; 4443;
16496; 16497; 22342; 22343; 22344; 22345; 22346; 22347; 22348;
3177; 2273; 22349; 22350; 22351; 22352; 22353; 22354; 22355; 22356;
22357; 22358; 10194; 3179; 3180; 3181; 3182; 3183; 3184; 3185;
3186; 22359; 22360; 22361; 3187; 22362; 3188; 10217; 10218; 16505;
6302; 22363; 22364; 10224; 22365; 22366; 22367; 22368; 22369;
22370; 22371; 579; 22372; 20379; 14685; 22373; 22374; 22375; 22376;
22377; 32227; 17277; 22378; 22379; 22380; 22381; 22382; 22383;
22384; 15669; 22385; 22386; 22387; 22388; 22389; 22390; 6311; 4464;
22391; 22392; 22393; 22394; 22395; 22396; 6313; 6314; 6315; 6316;
6317; 6318; 6319; 6320; 6321; 6322; 6323; 6324; 6325; 6326; 6327;
6328; 6329; 6330; 6331; 6332; 6333; 6334; 6335; 6336; 6337; 6338;
6339; 6340; 6341; 6342; 6343; 6344; 6345; 6346; 6347; 6348; 6349;
6350; 6351; 6352; 6353; 6354; 6355; 6356; 6357; 6358; 6359; 6360;
6361; 6362; 6363; 6364; 6365; 6366; 6367; 6368; 6369; 6370; 6371;
6372; 6373; 6374; 6375; 6376; 6377; 6378; 6379; 6380; 6381; 6382;
6383; 6384; 6385; 6386; 6387; 6388; 6389; 6390; 6391; 6392; 6393;
6394; 6395; 6396; 6397; 6398; 6399; 6400; 6401; 6402; 6403; 6404;
6405; 6406; 6407; 6408; 6409; 6410; 6411; 6412; 6413; 6414; 6415;
6416; 6417; 6418; 6419; 6420; 6421; 6422; 6423; 6424; 6425; 6426;
6427; 6428; 6429; 6430; 6431; 6432; 6433; 6434; 6435; 6436; 6437;
6438; 6439; 6440; 6441; 6442; 6443; 6444; 6445; 6446; 6447; 6448;
6449; 16521; 10244; 10245; 22397; 22398; 22399; 22400; 22401;
22402; 25564; 22403; 16530; 22404; 22405; 22406; 22407; 18289;
18290; 14707; 22408; 22409; 22410; 22411; 21012; 21013; 21014;
17283; 22412; 22413; 10287; 3215; 22414; 22415; 22416; 22417;
22418; 22419; 22420; 22421; 22422; 4497; 22423; 22424; 22425; 1484;
2295; 16538; 22426; 22427; 22428; 22429; 17289; 17290; 4501; 22430;
22431; 13023; 32228; 22432; 22433; 32229; 22434; 22435; 22436;
3229; 3230; 3231; 22437; 4513; 22438; 22439; 6516; 6517; 6518;
22440; 22441; 22442; 22443; 22444; 22445; 22446; 4521; 4522; 22447;
22448; 22449; 22450; 6520; 7766; 22451; 22452; 22453; 22454; 22455;
22456; 3237; 3238; 10389; 10390; 10391; 22457; 22458; 22459; 633;
634; 22460; 22461; 14757; 14758; 14759; 4535; 22462; 22463; 22464;
22465; 22466; 22467; 22468; 3247; 16565; 19622; 19623; 22469;
22470; 19624; 19625; 19626; 19627; 22471; 22472; 19628; 19629;
19630; 19631; 22473; 7782; 7783; 7784; 7785; 10425; 22474; 22475;
22476; 16567; 16568; 22477; 22478; 22479; 22480; 22481; 32230;
22482; 22483; 10447; 22484; 22485; 22486; 22487; 22488; 22489;
22490; 10467; 22491; 6573; 22492; 22493; 18357; 19645; 19647;
19648; 19649; 4565; 10484; 10485; 4566; 4567; 7798; 22494; 22495;
4570; 19660; 22496; 22497; 16575; 10494; 10495; 14777; 2338; 19665;
6587; 10507; 10508; 10509; 10510; 10511; 22498; 16585; 22499;
22500; 22501; 22502; 22503; 22504; 10515; 10548; 22505; 22506;
22507; 22508; 14786; 2353; 22509; 22510; 22511; 22512; 19684;
22513; 22514; 4584; 1541; 3267; 3268; 32231; 6611; 22515; 7813;
7814; 7815; 22516; 22517; 1545; 1546; 1547; 1548; 1549; 1550;
10592; 10593; 10594; 10595; 10596; 10597; 10598; 13757; 22518;
22519; 25594; 32232; 22520; 3278; 4600; 4601; 22521; 22522; 22523;
22524; 22525; 22526; 22527; 22528; 1553; 22529; 6623; 4610; 22530;
19703; 1560; 22531; 22532; 22533; 22534; 10636; 21080; 22535; 695;
22536; 22537; 22538; 22539; 22540; 22541; 22542; 22543; 22544;
22545; 22546; 4624; 22547; 16626; 32233; 22548; 22549; 22550;
22551; 22552; 704; 22553; 22554; 22555; 22556; 22557; 22558; 22559;
10654; 14824; 22560; 10657; 22561; 22562; 10660; 22563; 22564;
22565; 22566; 13065; 22567; 22568; 22569; 22570; 13792; 711; 22571;
22572; 21092; 16643; 22573; 15716; 15717; 7851; 22574; 2380; 22575;
13071; 22576; 22577; 22578; 13794; 13795; 22579; 16654; 21097;
22580; 22581; 22582; 19732; 19733; 19734; 22583; 21100; 22584;
22585; 22586; 22587; 32234; 22588; 18424; 18425; 18426; 22589;
22590; 22591; 22592; 22593; 729; 13077; 13078; 22594; 17338; 16687;
22595; 22596; 22597; 22598; 22599; 10744; 10745; 22600; 22601;
22602; 22603; 22604; 22605; 22606; 22607; 22608; 22609.
[0168] The following SEQ ID NOs correspond to the polynucleotides
encoding specific to male organ--Testis-specific proteins as
described in Table 37A identified using MPSS: 27423; 27424; 14038;
27425; 27426; 21443; 27427; 27428; 27429; 27430; 27431; 32260;
27432; 27433; 5088; 27434; 27435; 27436; 27437; 32261; 27438;
27439; 27440; 27441; 27442; 8160; 15913; 27443; 27444; 21460;
27445; 27446; 27447; 27448; 27449; 27450; 27451; 27452; 27453;
27454; 27455; 27456; 27457; 27458; 27459; 32262; 27460; 12678;
5109; 27461; 27462; 27463; 13276; 27464; 27465; 27466; 27467;
27468; 27469; 27470; 20586; 27471; 27472; 27473; 27474; 27475;
27476; 13277; 13278; 27477; 27478; 27479; 27; 28; 27480; 27481;
27482; 21483; 18967; 27483; 27484; 24735; 2653; 31; 32; 27485;
27486; 8228; 27487; 27488; 27489; 27490; 27491; 27492; 27493;
27494; 27495; 27496; 27497; 27498; 27499; 27500; 27501; 27502;
27503; 27504; 27505; 27506; 27507; 27508; 27509; 27510; 7246; 7247;
7248; 7249; 7250; 7251; 27511; 27512; 27513; 27514; 27515; 27516;
27517; 27518; 27519; 17531; 27520; 12689; 12690; 12691; 12692;
12693; 12694; 12695; 12696; 12697; 12698; 12699; 12700; 27521;
27522; 27523; 27524; 13304; 27525; 27526; 27527; 27528; 3646; 3647;
3648; 3649; 8248; 27529; 27530; 27531; 27532; 27533; 5135; 27534;
27535; 27536; 27537; 27538; 27539; 27540; 27541; 27542; 27543;
27544; 18981; 27545; 27546; 27547; 27548; 27549; 27550; 27551;
27552; 27553; 27554; 27555; 27556; 27557; 24745; 27558; 27559;
27560; 27561; 27562; 27563; 27564; 27565; 27566; 27567; 27568;
27569; 27570; 12709; 12710; 12711; 12712; 27571; 21517; 27572;
27573; 27574; 27575; 27576; 27577; 45; 20160; 27578; 47; 2677;
27579; 27580; 7271; 27581; 27582; 27583; 27584; 27585; 27586;
27587; 27588; 27589; 27590; 27591; 27592; 8275; 3677; 27593; 27594;
20164; 27595; 27596; 27597; 20602; 27598; 27599; 27600; 27601;
1075; 27602; 27603; 27604; 27605; 20603; 27606; 21525; 27607;
23315; 23316; 27608; 27609; 27610; 27611; 23318; 8309; 8310; 8311;
25892; 27612; 27613; 27614; 1081; 27615; 27616; 27617; 14109;
17579; 17580; 27618; 27619; 27620; 27621; 32263; 65; 27622; 27623;
27624; 27625; 27626; 27627; 27628; 27629; 27630; 19049; 19050;
27631; 27632; 27633; 27634; 27635; 27636; 27637; 8353; 27638;
27639; 8356; 27640; 27641; 19052; 23340; 27642; 27643; 27644;
27645; 93; 94; 27646; 11981; 27647; 27648; 27649; 27650; 27651;
27652; 27653; 21557; 21558; 21559; 21560; 21561; 7289; 27654;
27655; 27656; 27657; 27658; 1100; 27659; 13337; 27660; 27661;
27662; 27663; 27664; 27665; 27666; 27667; 27668; 27669; 27670;
27671; 20179; 20180; 20181; 27672; 27673; 27674; 27675; 27676;
27677; 21578; 8426; 27678; 1885; 21581; 27679; 8447; 27680; 27681;
27682; 27683; 8459; 17611; 1891; 27684; 20183; 20184; 27685; 27686;
27687; 27688; 27689; 7299; 27690; 5241; 27691; 27692; 27693; 27694;
27695; 27696; 27697; 27698; 13350; 27699; 27700; 27701; 27702;
27703; 27704; 27705; 27706; 27707; 27708; 27709; 27710; 27711;
23360; 27712; 27713; 27714; 27715; 27716; 27717; 27718; 27719;
27720; 27721; 1119; 8507; 27722; 27723; 25382; 27724; 27725; 27726;
27727; 27728; 27729; 27730; 27731; 27732; 27733; 27734; 27735;
21613; 27736; 27737; 27738; 27739; 27740; 27741; 27742; 27743;
27744; 27745; 27746; 27747; 27748; 27749; 27750; 27751; 27752;
21616; 27753; 27754; 27755; 27756; 27757; 27758; 25947; 25948;
27759; 27760; 27761; 27762; 27763; 27764; 8533; 13354; 27765;
27766; 27767; 27768; 27769; 27770; 19083; 19084; 13355; 27771;
7320; 7321; 27772; 27773; 27774; 27775; 126; 127; 128; 27776;
27777; 27778; 27779; 27780; 27781; 27782; 8559; 27783; 27784;
27785; 27786; 27787; 27788; 27789; 27790; 27791; 25959; 24802;
3765; 27792; 8573; 27793; 27794; 27795; 27796; 27797; 1142; 27798;
3774; 27799; 27800; 27801; 27802; 27803; 27804; 8605; 5292; 17083;
8608; 27805; 27806; 27807; 8613; 8614; 8615; 8616; 27808; 27809;
2743; 27810; 27811; 27812; 27813; 27814; 12009; 12010; 27815;
27816; 14202; 14203; 14204; 14205; 27817; 27818; 27819; 8646;
27820; 3791; 27821; 27822; 27823; 27824; 27825; 27826; 27827;
27828; 27829; 20678; 15458; 8653; 8654; 27830; 8658; 25401; 8661;
27831; 27832; 27833; 23402; 23403; 27834; 27835; 20683; 25402;
27836; 27837; 8677; 27838; 27839; 27840; 27841; 27842; 27843;
27844; 14213; 27845; 27846; 27847; 8693; 8694; 1152; 1153; 19121;
27848; 27849; 32264; 27850; 3827; 3828; 3829; 3830; 3831; 3832;
3833; 3834; 3835; 3836; 3837; 3838; 3839; 3840; 3841; 3842; 3843;
3844; 3845; 3846; 12020; 12021; 27851; 27852; 27853; 27854; 2783;
13385; 27855; 27856; 2784; 27857; 27858; 27859; 5390; 27860; 27861;
27862; 27863; 27864; 27865; 27866; 23411; 23412; 23413; 27867;
27868; 27869; 27870; 2792; 27871; 27872; 27873; 27874; 27875;
27876; 27877; 27878; 27879; 27880; 27881; 27882; 27883; 27884;
27885; 27886; 27887; 27888; 27889; 27890; 32265; 27891; 27892;
27893; 27894; 27895; 27896; 27897; 27898; 27899; 27900; 2794; 2795;
2796; 2797; 2798; 2799; 2800; 2801; 2802; 27901; 27902; 27903;
27904; 27905; 27906; 27907; 27908; 27909; 27910; 27911; 12031;
27912; 27913; 27914; 27915; 27916; 27917; 27918; 27919; 27920;
27921; 27922; 27923; 27924; 27925; 27926; 27927; 27928; 27929;
27930; 27931; 27932; 27933; 27934; 27935; 27936; 21700; 27937;
27938; 27939; 27940; 27941; 27942; 27943; 27944; 27945; 27946;
27947; 27948; 5434; 27949; 27950; 27951; 27952; 27953; 27954;
27955; 27956; 27957; 27958; 27959; 27960; 27961; 27962; 27963;
27964; 27965; 27966; 27967; 27968; 27969; 27970; 27971; 27972;
27973; 27974; 27975; 27976; 27977; 27978; 27979; 27980; 27981;
27982; 27983; 27984; 27985; 27986; 3874; 27987; 3877; 27988; 27989;
27990; 12036; 27991; 27992; 27993; 3880; 27994; 27995; 27996;
27997; 27998; 27999; 28000; 28001; 28002; 28003; 28004; 28005;
28006; 28007; 28008; 32266; 32267; 28009; 28010; 28011; 28012;
28013; 28014; 28015; 28016; 28017; 28018; 28019; 28020; 28021;
28022; 28023; 28024; 28025; 28026; 28027; 28028; 28029; 28030;
28031; 28032; 28033; 21723; 28034; 28035; 21724; 21725; 28036;
28037; 28038; 28039; 28040; 28041; 28042; 28043; 28044; 28045;
28046; 28047; 21726; 21727; 21728; 21729; 21730; 21731; 28048;
28049; 28050; 28051; 28052; 21732; 21733; 21734; 21735; 21736;
21737; 28053; 28054; 28055; 28056; 28057; 28058; 28059; 28060;
28061; 28062; 28063; 28064; 28065; 21742; 28066; 28067; 28068;
32268; 32269; 28069; 1164; 8838; 28070; 28071; 28072; 28073; 8844;
24852; 28074; 28075; 28076; 28077; 28078; 28079; 28080; 28081;
28082; 28083; 2816; 19161; 19162; 28084; 12050; 28085; 23457;
23458; 23459; 8862; 28086; 28087; 28088; 28089; 28090; 28091;
28092; 28093; 28094; 14261; 14262; 28095; 210; 28096; 28097; 12799;
28098; 28099; 26067; 28100; 28101; 28102; 28103; 12800; 28104;
28105; 28106; 8894; 5468; 28107; 21782; 28108; 28109; 28110; 14267;
28111; 28112; 28113; 28114; 28115; 28116; 28117; 25420; 5475;
28118; 32270; 28119; 28120; 17814; 28121; 3918; 217; 218; 219;
16125; 13428; 13429; 28122; 20239; 14289; 26095; 2848; 17820;
17821; 8945; 231; 232; 8951; 8952; 8953; 28123; 8957; 8958; 28124;
28125; 28126; 28127; 28128; 28129; 28130; 28131; 28132; 28133;
3937; 28134; 28135; 28136; 28137; 28138; 28139; 28140; 28141;
28142; 28143; 20749; 28144; 28145; 28146; 28147; 28148; 2861; 8970;
28149; 28150; 28151; 28152; 28153; 3948; 28154; 28155; 28156;
28157; 28158; 28159; 8980; 28160; 8981; 8982; 28161; 8983; 8984;
28162; 8985; 28163; 8986; 28164; 8987; 28165; 28166; 8988; 28167;
8989; 28168; 8990; 28169; 8991; 28170; 8992; 8993; 28171; 8994;
28172; 28173; 8995; 8996; 28174; 8997; 28175; 28176; 28177; 5659;
5660; 9001; 9002; 28178; 28179; 28180; 32271; 14317; 14318; 14319;
14320; 14321; 28181; 28182; 28183; 28184; 28185; 2871; 2872; 9010;
249; 250; 251; 28186; 28187; 28188; 28189; 28190; 28191; 28192;
15507; 28193; 28194; 16147; 28195; 28196; 28197; 21853; 21854;
21855; 21856; 21857; 21858; 21859; 13443; 28198; 28199; 28200;
28201; 28202; 28203; 5687; 13456; 28204; 28205; 28206; 28207;
28208; 28209; 28210; 28211; 28212; 28213; 28214; 28215; 28216;
28217; 28218; 28219; 28220; 28221; 28222; 2885; 28223; 28224;
28225; 28226; 2889; 28227; 28228; 9098; 23511; 23512; 23513; 23514;
23515; 28229; 21889; 271; 28230; 28231; 12100; 3995; 274; 278;
28232; 28233; 28234; 28235; 28236; 28237; 4008; 16175; 28238; 5733;
28239; 28240; 28241; 28242; 28243; 283; 12827; 284; 28244; 28245;
28246; 28247; 23530; 23531; 28248; 26161; 26162; 28249; 28250;
28251; 28252; 28253; 28254; 28255; 28256; 28257; 28258; 28259;
28260; 28261; 28262; 28263; 28264; 28265; 28266; 28267; 9148;
28268; 19250; 19251; 19252; 19255; 28269; 28270; 28271; 28272;
12832; 1241; 28273; 291; 28274; 5753; 28275; 17897; 28276; 28277;
2901; 28278; 28279; 28280; 28281; 28282; 1253; 1254; 28283; 28284;
9236; 28285; 28286; 28287; 21922; 21923; 25453; 28288; 28289;
28290; 28291; 28292; 28293; 28294; 28295; 26197; 1259; 1260; 1261;
7480; 7481; 28296; 28297; 28298; 24898; 16206; 9260; 28299; 28300;
305; 306; 307; 2058; 2059; 28301; 28302; 28303; 28304; 28305;
28306; 28307; 28308; 28309; 308; 309; 28310; 28311; 28312; 28313;
7486; 28314; 28315; 28316; 12129; 28317; 28318; 28319; 28320; 2920;
2921; 24900; 28321; 28322; 28323; 28324; 28325; 14394; 9291; 28326;
4061; 9297; 28327; 28328; 28329; 28330; 5790; 28331; 32272; 32273;
28332; 17921; 21946; 28333; 28334; 28335; 28336; 21957; 21958;
28337; 28338; 28339; 4076; 23571; 9320; 20818; 28340; 28341; 28342;
28343; 12141; 28344; 28345; 28346; 28347; 28348; 28349; 28350;
14409; 28351; 28352; 28353; 28354; 12148; 28355; 28356; 28357;
28358; 28359; 28360; 16238; 28361; 2080; 16245; 28362; 28363;
28364; 12857; 17174; 28365; 28366; 28367; 28368; 5820; 28369;
28370; 28371; 28372; 28373; 28374; 28375; 28376; 28377; 28378;
28379; 28380; 9349; 7521; 28381; 28382; 4100; 28383; 28384; 1299;
28385; 21991; 1300; 28386; 28387; 28388; 28389; 28390; 9365; 9366;
9367; 9368; 4106; 28391; 2963; 2964; 2965; 2966; 12151; 27359;
23595; 9380; 28392; 28393; 28394; 5842; 28395; 355; 5846; 5847;
5848; 22002; 28396; 28397; 28398; 28399; 9396; 9397; 5880; 28400;
28401; 361; 28402; 12155; 4112; 28403; 4120; 1307; 26267; 13529;
28404; 12874; 28405; 28406; 28407; 26273; 26274; 28408; 28409;
28410; 28411; 28412; 28413; 28414; 28415; 28416; 28417; 22021;
28418; 28419; 28420; 28421; 28422; 28423; 12876; 12877; 28424;
7541.
[0169] The following SEQ ID NOs correspond to the amino acid
sequences of specific to male organ--testis-specific proteins as
described in Table 37A identified using MPSS: 28425; 28426; 14452;
28427; 28428; 22030; 28429; 28430; 28431; 28432; 28433; 32274;
28434; 28435; 5922; 28436; 28437; 28438; 28439; 32275; 28440;
28441; 28442; 28443; 28444; 9488; 16310; 28445; 28446; 22047;
28447; 28448; 28449; 28450; 28451; 28452; 28453; 28454; 28455;
28456; 28457; 28458; 28459; 28460; 28461; 32276; 28462; 12886;
5943; 28463; 28464; 28465; 13543; 28466; 28467; 28468; 28469;
28470; 28471; 28472; 20853; 28473; 28474; 28475; 28476; 28477;
28478; 13544; 13545; 28479; 28480; 28481; 395; 396; 28482; 28483;
28484; 22070; 19371; 28485; 28486; 24938; 3009; 399; 400; 28487;
28488; 9556; 28489; 28490; 28491; 28492; 28493; 28494; 28495;
28496; 28497; 28498; 28499; 28500; 28501; 28502; 28503; 28504;
28505; 28506; 28507; 28508; 28509; 28510; 28511; 28512; 7576; 7577;
7578; 7579; 7580; 7581; 28513; 28514; 28515; 28516; 28517; 28518;
28519; 28520; 28521; 18000; 28522; 12897; 12898; 12899; 12900;
12901; 12902; 12903; 12904; 12905; 12906; 12907; 12908; 28523;
28524; 28525; 28526; 13571; 28527; 28528; 28529; 28530; 4195; 4196;
4197; 4198; 9576; 28531; 28532; 28533; 28534; 28535; 5969; 28536;
28537; 28538; 28539; 28540; 28541; 28542; 28543; 28544; 28545;
28546; 19385; 28547; 28548; 28549; 28550; 28551; 28552; 28553;
28554; 28555; 28556; 28557; 28558; 28559; 24948; 28560; 28561;
28562; 28563; 28564; 28565; 28566; 28567; 28568; 28569; 28570;
28571; 28572; 12917; 12918; 12919; 12920; 28573; 22104; 28574;
28575; 28576; 28577; 28578; 28579; 413; 20301; 28580; 415; 3033;
28581; 28582; 7601; 28583; 28584; 28585; 28586; 28587; 28588;
28589; 28590; 28591; 28592; 28593; 28594; 9603; 4226; 28595; 28596;
20305; 28597; 28598; 28599; 20869; 28600; 28601; 28602; 28603;
1358; 28604; 28605; 28606; 28607; 20870; 28608; 22112; 28609;
23646; 23647; 28610; 28611; 28612; 28613; 23649; 9637; 9638; 9639;
26362; 28614; 28615; 28616; 1364; 28617; 28618; 28619; 14523;
18048; 18049; 28620; 28621; 28622; 28623; 32277; 433; 28624; 28625;
28626; 28627; 28628; 28629; 28630; 28631; 28632; 19453; 19454;
28633; 28634; 28635; 28636; 28637; 28638; 28639; 9681; 28640;
28641; 9684; 28642; 28643; 19456; 23671; 28644; 28645; 28646;
28647; 461; 462; 28648; 12237; 28649; 28650; 28651; 28652; 28653;
28654; 28655; 22144; 22145; 22146; 22147; 22148; 7619; 28656;
28657; 28658; 28659; 28660; 1383; 28661; 13604; 28662; 28663;
28664; 28665; 28666; 28667; 28668; 28669; 28670; 28671; 28672;
28673; 20320; 20321; 20322; 28674; 28675; 28676; 28677; 28678;
28679; 22165; 9754; 28680; 2184; 22168; 28681; 9775; 28682; 28683;
28684; 28685; 9787; 18080; 2190; 28686; 20324; 20325; 28687; 28688;
28689; 28690; 28691; 7629; 28692; 6075; 28693; 28694; 28695; 28696;
28697; 28698; 28699; 28700; 13617; 28701; 28702; 28703; 28704;
28705; 28706; 28707; 28708; 28709; 28710; 28711; 28712; 28713;
23691; 28714; 28715; 28716; 28717; 28718; 28719; 28720; 28721;
28722; 28723; 1402; 9835; 28724; 28725; 25522; 28726; 28727; 28728;
28729; 28730; 28731; 28732; 28733; 28734; 28735; 28736; 28737;
22200; 28738; 28739; 28740; 28741; 28742; 28743; 28744; 28745;
28746; 28747; 28748; 28749; 28750; 28751; 28752; 28753; 28754;
22203; 28755; 28756; 28757; 28758; 28759; 28760; 26417; 26418;
28761; 28762; 28763; 28764; 28765; 28766; 9861; 13621; 28767;
28768; 28769; 28770; 28771; 28772; 19487; 19488; 13622; 28773;
7650; 7651; 28774; 28775; 28776; 28777; 494; 495; 496; 28778;
28779; 28780; 28781; 28782; 28783; 28784; 9887; 28785; 28786;
28787; 28788; 28789; 28790; 28791; 28792; 28793; 26429; 25005;
4314; 28794; 9901; 28795; 28796; 28797; 28798; 28799; 1425; 28800;
4323; 28801; 28802; 28803; 28804; 28805; 28806; 9933; 6126; 17243;
9936; 28807; 28808; 28809; 9941; 9942; 9943; 9944; 28810; 28811;
3099; 28812; 28813; 28814; 28815; 28816; 12265; 12266; 28817;
28818; 14616; 14617; 14618; 14619; 28819; 28820; 28821; 9974;
28822; 4340; 28823; 28824; 28825; 28826; 28827; 28828; 28829;
28830; 28831; 20945; 15634; 9981; 9982; 28832; 9986; 25541; 9989;
28833; 28834; 28835; 23733; 23734; 28836; 28837; 20950; 25542;
28838; 28839; 10005; 28840; 28841; 28842; 28843; 28844; 28845;
28846; 14627; 28847; 28848; 28849; 10021; 10022; 1435; 1436; 19525;
28850; 28851; 32278; 28852; 4376; 4377; 4378; 4379; 4380; 4381;
4382; 4383; 4384; 4385; 4386; 4387; 4388; 4389; 4390; 4391; 4392;
4393; 4394; 4395; 12276; 12277; 28853; 28854; 28855; 28856; 3139;
13652; 28857; 28858; 3140; 28859; 28860; 28861; 6224; 28862; 28863;
28864; 28865; 28866; 28867; 28868; 23742; 23743; 23744; 28869;
28870; 28871; 28872; 3148; 28873; 28874; 28875; 28876; 28877;
28878; 28879; 28880; 28881; 28882; 28883; 28884; 28885; 28886;
28887; 28888; 28889; 28890; 28891; 28892; 32279; 28893; 28894;
28895; 28896; 28897; 28898; 28899; 28900; 28901; 28902; 3150; 3151;
3152; 3153; 3154; 3155; 3156; 3157; 3158; 28903; 28904; 28905;
28906; 28907; 28908; 28909; 28910; 28911; 28912; 28913; 12287;
28914; 28915; 28916; 28917; 28918; 28919; 28920; 28921; 28922;
28923; 28924; 28925; 28926; 28927; 28928; 28929; 28930; 28931;
28932; 28933; 28934; 28935; 28936; 28937; 28938; 22287; 28939;
28940; 28941; 28942; 28943; 28944; 28945; 28946; 28947; 28948;
28949; 28950; 6268; 28951; 28952; 28953; 28954; 28955; 28956;
28957; 28958; 28959; 28960; 28961; 28962; 28963; 28964; 28965;
28966; 28967; 28968; 28969; 28970; 28971; 28972; 28973; 28974;
28975; 28976; 28977; 28978; 28979; 28980; 28981; 28982; 28983;
28984; 28985; 28986; 28987; 28988; 4423; 28989; 4426; 28990; 28991;
28992; 12292; 28993; 28994; 28995; 4429; 28996; 28997; 28998;
28999; 29000; 29001; 29002; 29003; 29004; 29005; 29006; 29007;
29008; 29009; 29010; 32280; 32281; 29011; 29012; 29013; 29014;
29015; 29016; 29017; 29018; 29019; 29020; 29021; 29022; 29023;
29024; 29025; 29026; 29027; 29028; 29029; 29030; 29031; 29032;
29033; 29034; 29035; 22310; 29036; 29037; 22311; 22312; 29038;
29039; 29040; 29041; 29042; 29043; 29044; 29045; 29046; 29047;
29048; 29049; 22313; 22314; 22315; 22316; 22317; 22318; 29050;
29051; 29052; 29053; 29054; 22319; 22320; 22321; 22322; 22323;
22324; 29055; 29056; 29057; 29058; 29059; 29060; 29061; 29062;
29063; 29064; 29065; 29066; 29067; 22329; 29068; 29069; 29070;
32282; 32283; 29071; 1447; 10166; 29072; 29073; 29074; 29075;
10172; 25055; 29076; 29077; 29078; 29079; 29080; 29081; 29082;
29083; 29084; 29085; 3172; 19565; 19566; 29086; 12306; 29087;
23788; 23789; 23790; 10190; 29088; 29089; 29090; 29091; 29092;
29093; 29094; 29095; 29096; 14675; 14676; 29097; 578; 29098; 29099;
13007; 29100; 29101; 26537; 29102; 29103; 29104; 29105; 13008;
29106; 29107; 29108; 10222; 6302; 29109; 22369; 29110; 29111;
29112; 14681; 29113; 29114; 29115; 29116; 29117; 29118; 29119;
25560; 6309; 29120; 32284; 29121; 29122; 18283; 29123; 4467; 585;
586; 587; 16522; 13695; 13696; 29124; 20380; 14703; 26565; 3204;
18289; 18290; 10273; 599; 600; 10279; 10280; 10281; 29125; 10285;
10286; 29126; 29127; 29128; 29129; 29130; 29131; 29132; 29133;
29134; 29135; 4486; 29136; 29137; 29138; 29139; 29140; 29141;
29142; 29143; 29144; 29145; 21016; 29146; 29147; 29148; 29149;
29150; 3217; 10298; 29151; 29152; 29153; 29154; 29155; 4497; 29156;
29157; 29158; 29159; 29160; 29161; 10308; 29162; 10309; 10310;
29163; 10311; 10312; 29164; 10313; 29165; 10314; 29166; 10315;
29167; 29168; 10316; 29169; 10317; 29170; 10318; 29171; 10319;
29172; 10320; 10321; 29173; 10322; 29174; 29175; 10323; 10324;
29176; 10325; 29177; 29178; 29179; 6493; 6494; 10329; 10330; 29180;
29181; 29182; 32285; 14731; 14732; 14733; 14734; 14735; 29183;
29184; 29185; 29186; 29187; 3227; 3228; 10338; 617; 618; 619;
29188; 29189; 29190; 29191; 29192; 29193; 29194; 15683; 29195;
29196; 16544; 29197; 29198; 29199; 22440; 22441; 22442; 22443;
22444; 22445; 22446; 13710; 29200; 29201; 29202; 29203; 29204;
29205; 6521; 13723; 29206; 29207; 29208; 29209; 29210; 29211;
29212; 29213; 29214; 29215; 29216; 29217; 29218; 29219; 29220;
29221; 29222; 29223; 29224; 3241; 29225; 29226; 29227; 29228; 3245;
29229; 29230; 10426; 23842; 23843; 23844; 23845; 23846; 29231;
22476; 639; 29232; 29233; 12356; 4544; 642; 646; 29234; 29235;
29236; 29237; 29238; 29239; 4557; 16572; 29240; 6567; 29241; 29242;
29243; 29244; 29245; 651; 13035; 652; 29246; 29247; 29248; 29249;
23861; 23862; 29250; 26631; 26632; 29251; 29252; 29253; 29254;
29255; 29256; 29257; 29258; 29259; 29260; 29261; 29262; 29263;
29264; 29265; 29266; 29267; 29268; 29269; 10476; 29270; 19654;
19655; 19656; 19659; 29271; 29272; 29273; 29274; 13040; 1524;
29275; 659; 29276; 6587; 29277; 18366; 29278; 29279; 3257; 29280;
29281; 29282; 29283; 29284; 1536; 1537; 29285; 29286; 10564; 29287;
29288; 29289; 22509; 22510; 25593; 29290; 29291; 29292; 29293;
29294; 29295; 29296; 29297; 26667; 1542; 1543; 1544; 7810; 7811;
29298; 29299; 29300; 25101; 16603; 10588; 29301; 29302; 673; 674;
675; 2357; 2358; 29303; 29304; 29305; 29306; 29307; 29308; 29309;
29310; 29311; 676; 677; 29312; 29313; 29314; 29315; 7816; 29316;
29317; 29318; 12385; 29319; 29320; 29321; 29322; 3276; 3277; 25103;
29323; 29324; 29325; 29326; 29327; 14808; 10619; 29328; 4610;
10625; 29329; 29330; 29331; 29332; 6624; 29333; 32286; 32287;
29334; 18390; 22533; 29335; 29336; 29337; 29338; 22544; 22545;
29339; 29340; 29341; 4625; 23902; 10648; 21085; 29342; 29343;
29344; 29345; 12397; 29346; 29347; 29348; 29349; 29350; 29351;
29352; 14823; 29353; 29354; 29355; 29356; 12404; 29357; 29358;
29359; 29360; 29361; 29362; 16635; 29363; 2379; 16642; 29364;
29365; 29366; 13065; 17334; 29367; 29368; 29369; 29370; 6654;
29371; 29372; 29373; 29374; 29375; 29376; 29377; 29378; 29379;
29380; 29381; 29382; 10677; 7851; 29383; 29384; 4649; 29385; 29386;
1582; 29387; 22578; 1583; 29388; 29389; 29390; 29391; 29392; 10693;
10694; 10695; 10696; 4655; 29393; 3319; 3320; 3321; 3322; 12407;
27389; 23926; 10708; 29394; 29395; 29396; 6676; 29397; 723; 6680;
6681; 6682; 22589; 29398; 29399; 29400; 29401; 10724; 10725; 6714;
29402; 29403; 729; 29404; 12411; 4661; 29405; 4669; 1590; 26737;
13796; 29406; 13082; 29407; 29408; 29409; 26743; 26744; 29410;
29411; 29412; 29413; 29414; 29415; 29416; 29417; 29418; 29419;
22608; 29420; 29421; 29422; 29423; 29424; 29425; 13084; 13085;
29426; 7871.
[0170] The following SEQ ID NOs correspond to the polynucleotides
encoding specific to Female Organ--Mammary Gland-specific proteins
as described in Table 38A identified using MPSS: 17023; 17024;
17025; 17026; 32311; 8155; 8156; 17027; 17028; 7228; 17029; 17030;
17031; 5101; 17032; 23288; 5107; 5108; 17033; 17034; 17035; 17036;
17037; 17038; 32312; 32313; 32314; 32315; 32316; 32317; 17039;
8210; 8211; 8212; 8213; 17040; 17041; 17042; 17043; 17044; 17045;
17046; 40; 17047; 2669; 17048; 17049; 17050; 17051; 17052; 17053;
17054; 32318; 17055; 17056; 13327; 17057; 17058; 17059; 17060;
2699; 2700; 2701; 2702; 17061; 17062; 17063; 17064; 32319; 8458;
17065; 17066; 17067; 17068; 17069; 17070; 17071; 17072; 17073;
17074; 17075; 17076; 17077; 17078; 17079; 32320; 5254; 5255; 5256;
5257; 5258; 5259; 5260; 32321; 27774; 17080; 17081; 131; 11994;
11995; 27793; 17082; 17083; 17084; 17085; 150; 151; 17086; 17087;
17088; 164; 17089; 17090; 17091; 17092; 17093; 17094; 17095; 17096;
17097; 17098; 17099; 17100; 17101; 17102; 17103; 17104; 17105;
17106; 17107; 17108; 17109; 17110; 24820; 17111; 32322; 32323;
14246; 2814; 2815; 3903; 17112; 17113; 17114; 8888; 17115; 2835;
17116; 17117; 17118; 17119; 17120; 17121; 17122; 17123; 32324;
17124; 28123; 17125; 14298; 17126; 8970; 17127; 17128; 15503;
17129; 17130; 32215; 17131; 17132; 17133; 17134; 17135; 17136;
17137; 17138; 17139; 17140; 32325; 32326; 32327; 32328; 17141;
17142; 17143; 17144; 17145; 17146; 17147; 17148; 17149; 17150;
1236; 13473; 9158; 16177; 9163; 17151; 17152; 17153; 17154; 17155;
17156; 17157; 14365; 2043; 2044; 2045; 2046; 17158; 17159; 16190;
17160; 17161; 4033; 9243; 17162; 5776; 17163; 12843; 16225; 17164;
7509; 17165; 14407; 17166; 17167; 17168; 1281; 17169; 17170; 9327;
17171; 17172; 14421; 17173; 7516; 12857; 17174; 17175; 1299; 12151;
5837; 5838; 17176; 355; 17177; 17178; 16293; 17179; 5892; 5895;
17180; 17181; 17182.
[0171] The following SEQ ID NOs correspond to the amino acid
sequences of specific to Female Organ--Mammary Gland-specific
proteins as described in Table 38A identified using MPSS: 17183;
17184; 17185; 17186; 32329; 9483; 9484; 17187; 17188; 7558; 17189;
17190; 17191; 5935; 17192; 23619; 5941; 5942; 17193; 17194; 17195;
17196; 17197; 17198; 32330; 32331; 32332; 32333; 32334; 32335;
17199; 9538; 9539; 9540; 9541; 17200; 17201; 17202; 17203; 17204;
17205; 17206; 408; 17207; 3025; 17208; 17209; 17210; 17211; 17212;
17213; 17214; 32336; 17215; 17216; 13594; 17217; 17218; 17219;
17220; 3055; 3056; 3057; 3058; 17221; 17222; 17223; 17224; 32337;
9786; 17225; 17226; 17227; 17228; 17229; 17230; 17231; 17232;
17233; 17234; 17235; 17236; 17237; 17238; 17239; 32338; 6088; 6089;
6090; 6091; 6092; 6093; 6094; 32339; 28776; 17240; 17241; 499;
12250; 12251; 28795; 17242; 17243; 17244; 17245; 518; 519; 17246;
17247; 17248; 532; 17249; 17250; 17251; 17252; 17253; 17254; 17255;
17256; 17257; 17258; 17259; 17260; 17261; 17262; 17263; 17264;
17265; 17266; 17267; 17268; 17269; 17270; 25023; 17271; 32340;
32341; 14660; 3170; 3171; 4452; 17272; 17273; 17274; 10216; 17275;
3191; 17276; 17277; 17278; 17279; 17280; 17281; 17282; 17283;
32342; 17284; 29125; 17285; 14712; 17286; 10298; 17287; 17288;
15679; 17289; 17290; 32229; 17291; 17292; 17293; 17294; 17295;
17296; 17297; 17298; 17299; 17300; 32343; 32344; 32345; 32346;
17301; 17302; 17303; 17304; 17305; 17306; 17307; 17308; 17309;
17310; 1519; 13740; 10486; 16574; 10491; 17311; 17312; 17313;
17314; 17315; 17316; 17317; 14779; 2342; 2343; 2344; 2345; 17318;
17319; 16587; 17320; 17321; 4582; 10571; 17322; 6610; 17323; 13051;
16622; 17324; 7839; 17325; 14821; 17326; 17327; 17328; 1564; 17329;
17330; 10655; 17331; 17332; 14835; 17333; 7846; 13065; 17334;
17335; 1582; 12407; 6671; 6672; 17336; 723; 17337; 17338; 16690;
17339; 6726; 6729; 17340; 17341; 17342.
[0172] The following SEQ ID NOs correspond to the polynucleotides
encoding specific to Female Organ--Uterus-specific proteins as
described in Table 39A identified using MPSS: 1031; 32003; 32004;
5091; 32005; 32006; 32007; 32008; 32009; 32010; 32011; 32012;
21481; 20149; 32013; 32014; 13305; 32015; 32016; 8280; 11961;
32017; 32018; 32019; 25906; 32020; 32021; 32022; 32023; 7309;
32024; 32025; 21631; 32026; 8603; 20656; 32027; 32028; 21646;
32029; 32030; 8671; 20682; 32031; 32032; 8715; 8716; 32033; 5400;
32034; 32035; 32036; 32037; 32038; 32039; 32040; 16098; 21755;
21756; 32041; 32042; 32043; 8942; 8943; 8944; 32044; 21828; 32045;
32367; 32046; 32047; 32048; 32049; 32050; 32051; 19202; 1211; 9018;
9019; 32052; 32053; 32054; 32055; 32056; 32057; 23503; 23504;
31459; 31460; 31461; 31462; 31463; 31464; 31465; 31466; 31467;
31468; 32058; 20784; 32059; 23532; 23533; 23534; 23535; 23536;
13473; 19269; 19270; 32060; 32061; 21921; 26197; 2917; 2918; 20813;
32062; 31484; 1285; 1286; 1287; 1288; 1289; 1290; 1291; 27357;
32063; 5898; 366; 9426; 9427; 9432; 9433; 32064; 32065;
[0173] The following SEQ ID NOs correspond to the amino acid
sequences of specific to Female Organ--Uterus-specific proteins as
described in Table 39A identified using MPSS: 1314; 32066; 32067;
5925; 32068; 32069; 32070; 32071; 32072; 32073; 32074; 32075;
22068; 20290; 32076; 32077; 13572; 32078; 32079; 9608; 12217;
32080; 32081; 32082; 26376; 32083; 32084; 32085; 32086; 7639;
32087; 32088; 22218; 32089; 9931; 20923; 32090; 32091; 22233;
32092; 32093; 9999; 20949; 32094; 32095; 10043; 10044; 32096; 6234;
32097; 32098; 32099; 32100; 32101; 32102; 32103; 16495; 22342;
22343; 32104; 32105; 32106; 10270; 10271; 10272; 32107; 22415;
32108; 32368; 32109; 32110; 32111; 32112; 32113; 32114; 19606;
1494; 10346; 10347; 32115; 32116; 32117; 32118; 32119; 32120;
23834; 23835; 31621; 31622; 31623; 31624; 31625; 31626; 31627;
31628; 31629; 31630; 32121; 21051; 32122; 23863; 23864; 23865;
23866; 23867; 13740; 19673; 19674; 32123; 32124; 22508; 26667;
3273; 3274; 21080; 32125; 31646; 1568; 1569; 1570; 1571; 1572;
1573; 1574; 27387; 32126; 6732; 734; 10754; 10755; 10760; 10761;
32127; 32128.
[0174] The following SEQ ID NOs correspond to the polynucleotides
encoding CL1 prostate cancer cell-specific proteins as described in
Table 40A identified using MPSS: 32374; 17023; 32375; 32376; 12;
13; 32377; 32378; 5110; 5111; 32379; 8199; 8203; 8204; 8205; 8206;
32380; 5127; 3629; 32381; 32382; 32383; 32384; 32385; 1826; 32386;
32387; 2663; 2664; 32388; 11950; 7257; 32389; 25869; 20596; 32390;
32391; 32392; 32393; 21522; 32394; 32395; 32396; 32397; 32398;
15416; 32399; 11965; 11966; 32400; 14109; 32401; 25369; 8362; 8363;
32402; 20617; 7290; 32403; 32404; 32405; 101; 32406; 1105; 1106;
32407; 7299; 14145; 32408; 30506; 21592; 2717; 32409; 32410; 32411;
32412; 32413; 1124; 32414; 32415; 13363; 32416; 32417; 32418;
32419; 32420; 32421; 25973; 5301; 7346; 20211; 32422; 32423; 32424;
27831; 30542; 32425; 32426; 19120; 27853; 30551; 23411; 23412;
23413; 7360; 32427; 20220; 13394; 32428; 13397; 32429; 31873;
30603; 30606; 19143; 32430; 5450; 21755; 21756; 8858; 21762; 21763;
21764; 21765; 21766; 21767; 21768; 8869; 28092; 28093; 32431; 1174;
1175; 1176; 1177; 1178; 32432; 32433; 32434; 32435; 1179; 1180;
1181; 1182; 1183; 1184; 32436; 5471; 23463; 32437; 32438; 26079;
26081; 26082; 26084; 26085; 26087; 26088; 26090; 13418; 13419;
32439; 3925; 32440; 26104; 26105; 32441; 32442; 12811; 12812;
21832; 21833; 21834; 32443; 32444; 28177; 32445; 14317; 14318;
14319; 14320; 14321; 32214; 32446; 5667; 32447; 32448; 32449;
32450; 32451; 26134; 26135; 32452; 32453; 13458; 13459; 13460;
13461; 13462; 13463; 2890; 32454; 2038; 32455; 32456; 28274; 19264;
23545; 23546; 32457; 7474; 32458; 32459; 14376; 16200; 32460;
21933; 310; 20801; 32461; 9315; 2073; 32462; 331; 12850; 17930;
32463; 32464; 32465; 4089; 5819; 32466; 1297; 32467; 32468; 32469;
32470; 32471; 32472; 22002; 32473; 32474; 12874; 32475; 32476;
12875; 26277; 32477; 32478; 32479; 32480.
[0175] The following SEQ ID NOs correspond to the amino acid
sequences of CL1 prostate cancer cell-specific proteins as
described in Table 40A identified using MPSS: 32481; 17183; 32482;
32483; 380; 381; 32484; 32485; 5944; 5945; 32486; 9527; 9531; 9532;
9533; 9534; 32487; 5961; 4178; 32488; 32489; 32490; 32491; 32492;
2125; 32493; 32494; 3019; 3020; 32495; 12206; 7587; 32496; 26339;
20863; 32497; 32498; 32499; 32500; 22109; 32501; 32502; 32503;
32504; 32505; 15592; 32506; 12221; 12222; 32507; 14523; 32508;
25509; 9690; 9691; 32509; 20884; 7620; 32510; 32511; 32512; 469;
32513; 1388; 1389; 32514; 7629; 14559; 32515; 30769; 22179; 3073;
32516; 32517; 32518; 32519; 32520; 1407; 32521; 32522; 13630;
32523; 32524; 32525; 32526; 32527; 32528; 26443; 6135; 7676; 20352;
32529; 32530; 32531; 28833; 30805; 32532; 32533; 19524; 28855;
30814; 23742; 23743; 23744; 7690; 32534; 20361; 13661; 32535;
13664; 32536; 31915; 30866; 30869; 19547; 32537; 6284; 22342;
22343; 10186; 22349; 22350; 22351; 22352; 22353; 22354; 22355;
10197; 29094; 29095; 32538; 1457; 1458; 1459; 1460; 1461; 32539;
32540; 32541; 32542; 1462; 1463; 1464; 1465; 1466; 1467; 32543;
6305; 23794; 32544; 32545; 26549; 26551; 26552; 26554; 26555;
26557; 26558; 26560; 13685; 13686; 32546; 4474; 32547; 26574;
26575; 32548; 32549; 13019; 13020; 22419; 22420; 22421; 32550;
32551; 29179; 32552; 14731; 14732; 14733; 14734; 14735; 32228;
32553; 6501; 32554; 32555; 32556; 32557; 32558; 26604; 26605;
32559; 32560; 13725; 13726; 13727; 13728; 13729; 13730; 3246;
32561; 2337; 32562; 32563; 29276; 19668; 23876; 23877; 32564; 7804;
32565; 32566; 14790; 16597; 32567; 22520; 678; 21068; 32568; 10643;
2372; 32569; 699; 13058; 18399; 32570; 32571; 32572; 4638; 6653;
32573; 1580; 32574; 32575; 32576; 32577; 32578; 32579; 22589;
32580; 32581; 13082; 32582; 32583; 13083; 26747; 32584; 32585;
32586; 32587.
[0176] The following SEQ ID NOs correspond to the polynucleotides
encoding LNCaP prostate cancer cell-specific proteins as described
in Table 41A identified using MPSS: 32757; 32758; 32759; 32760;
21467; 32761; 32762; 32763; 32764; 23296; 23297; 23298; 32765;
30482; 32766; 32767; 58; 59; 60; 5196; 32768; 32769; 32770; 32771;
1107; 1108; 5241; 30506; 20629; 8513; 8514; 30522; 27760; 25387;
19096; 131; 32772; 32773; 32774; 32775; 2740; 8602; 32776; 19108;
32777; 8632; 1149; 32778; 32779; 32780; 5358; 5408; 5409; 32781;
32782; 32783; 32784; 32785; 32786; 32787; 32788; 32789; 28085;
8872; 8873; 8874; 8875; 8876; 8877; 8878; 8879; 32790; 32791;
32792; 32793; 32794; 32795; 21829; 23485; 1997; 1998; 32796; 14312;
30648; 14313; 14314; 14315; 32797; 32798; 32799; 32800; 32801;
5662; 1210; 2873; 2874; 2875; 32802; 21860; 32803; 32804; 21870;
14350; 32805; 32806; 32807; 32218; 32808; 21938; 32809; 32810;
32811; 32812; 5887.
[0177] The following SEQ ID NOs correspond to the amino acid
sequences of LNCaP prostate cancer cell-specific proteins as
described in Table 41A identified using MPSS: 32813; 32814; 32815;
32816; 22054; 32817; 32818; 32819; 32820; 23627; 23628; 23629;
32821; 30745; 32822; 32823; 426; 427; 428; 6030; 32824; 32825;
32826; 32827; 1390; 1391; 6075; 30769; 20896; 9841; 9842; 30785;
28762; 25527; 19500; 499; 32828; 32829; 32830; 32831; 3096; 9930;
32832; 19512; 32833; 9960; 1432; 32834; 32835; 32836; 6192; 6242;
6243; 32837; 32838; 32839; 32840; 32841; 32842; 32843; 32844;
32845; 29087; 10200; 10201; 10202; 10203; 10204; 10205; 10206;
10207; 32846; 32847; 32848; 32849; 32850; 32851; 22416; 23816;
2296; 2297; 32852; 14726; 30911; 14727; 14728; 14729; 32853; 32854;
32855; 32856; 32857; 6496; 1493; 3229; 3230; 3231; 32858; 22447;
32859; 32860; 22457; 14764; 32861; 32862; 32863; 32232; 32864;
22525; 32865; 32866; 32867; 32868; 6721.
[0178] The following SEQ ID NOs correspond to the polynucleotides
encoding male organ, prostate-specific proteins identified using
MPSS as described in Table 42A and Example 7: 21436; 21437; 15907;
21438; 21439; 21440; 21441; 3582; 3583; 3584; 3585; 21442; 13270;
8131; 21443; 1801; 1032; 8135; 14042; 11908; 11909; 11910; 21444;
21445; 21446; 21447; 8144; 8145; 21448; 21449; 21450; 21451; 21452;
21453; 21454; 21455; 21456; 21457; 21458; 2633; 2634; 2635; 21459;
21460; 21461; 15; 1040; 16; 5101; 21462; 21463; 21464; 21465;
21466; 21467; 21468; 5102; 1814; 1041; 1042; 1043; 21469; 2638;
2639; 1044; 21470; 21471; 21472; 8187; 21473; 21474; 21475; 21476;
21477; 5110; 5111; 3622; 3623; 21478; 2650; 8198; 15403; 1820;
5122; 5123; 5124; 5125; 8200; 21479; 5126; 1047; 17520; 21480;
21482; 7238; 29; 21483; 21484; 8226; 21485; 21486; 21487; 21488;
21489; 21490; 32208; 21491; 21492; 21493; 21494; 21495; 21496;
21497; 21498; 21499; 21500; 21501; 18976; 18977; 21502; 21503;
15937; 21504; 1833; 21505; 21506; 21507; 21508; 5145; 21509; 21510;
11951; 21511; 21512; 21513; 15943; 21514; 18983; 18984; 18985;
18986; 18987; 18988; 18989; 18990; 5156; 5157; 21515; 21516; 21517;
21518; 3670; 21520; 21521; 19008; 21522; 1074; 20603; 19015; 56;
21523; 21524; 21525; 21526; 8300; 21527; 21528; 21529; 21530;
14100; 14101; 21531; 19017; 19018; 32209; 21532; 21533; 21534;
20605; 21535; 21536; 11967; 21537; 14107; 21538; 21539; 8326;
21540; 1869; 1870; 19043; 19044; 19045; 21541; 21542; 21543; 21544;
7282; 15423; 16001; 21546; 15428; 21547; 21548; 8356; 17589; 21549;
7283; 21550; 21551; 7285; 21553; 21554; 90; 8366; 8367; 21555;
21556; 21557; 21558; 21559; 21560; 21561; 21562; 21563; 20618;
20619; 21564; 21565; 21566; 21567; 21568; 8408; 5220; 21569; 21570;
21571; 11982; 2699; 21572; 21573; 21574; 1101; 21575; 21576; 1103;
101; 8422; 8423; 21577; 21578; 21579; 21580; 1104; 1885; 21581;
15441; 2705; 21582; 21583; 8458; 14141; 14142; 21584; 21585; 21586;
20622; 20623; 21587; 21588; 21589; 17065; 17066; 17067; 5242; 5243;
8470; 8471; 8476; 8477; 21590; 21591; 21592; 14147; 21593; 1897;
21594; 21595; 21596; 21597; 21598; 21599; 21600; 21601; 21602;
21603; 7308; 7309; 8508; 3738; 17620; 21605; 21606; 21607; 21608;
21609; 21610; 21611; 21612; 21613; 21614; 21615; 19078; 21616;
21617; 19082; 121; 122; 123; 124; 5254; 5256; 5257; 5258; 5259;
5260; 21618; 1122; 1123; 21619; 21620; 21621; 8547; 21622; 17636;
17637; 21623; 21624; 8551; 8552; 1132; 21625; 21626; 2730; 2731;
131; 132; 13364; 21627; 21628; 21629; 21630; 21631; 21632; 21633;
21634; 21635; 21636; 21637; 21638; 21639; 7339; 137; 8596; 8597;
8600; 21640; 1142; 21641; 21642; 21643; 14188; 21645; 21646; 21647;
5301; 7346; 21648; 21649; 21650; 156; 21651; 1938; 8641; 8642;
8643; 8644; 1939; 21652; 3796; 3797; 3798; 3799; 3800; 3801; 21653;
21654; 8666; 8667; 21655; 2753; 8674; 3821; 2755; 21656; 15459;
5359; 5360; 5361; 5362; 5363; 5364; 5365; 5366; 5367; 5368; 5369;
5370; 5371; 5372; 5373; 5374; 5375; 21657; 21658; 21659; 8700;
14215; 14216; 14217; 14219; 14220; 14222; 21660; 21661; 21662;
2782; 21663; 21664; 21665; 21666; 21667; 21668; 21669; 21670;
21671; 21672; 21673; 21674; 21675; 21676; 21677; 20698; 21678;
21679; 21680; 2792; 5397; 5398; 8735; 8736; 8737; 8738; 8739; 8740;
8741; 8742; 21681; 21682; 21683; 21684; 21685; 21686; 21687; 21688;
21689; 21690; 21691; 21692; 21693; 21694; 32210; 21695; 21696;
21697; 21698; 3867; 21699; 21700; 21701; 187; 21703; 21704; 21705;
21706; 21707; 21708; 21710; 21711; 21713; 21714; 21715; 21716;
32211; 21717; 21718; 21719; 21721; 21722; 21723; 21724; 21725;
21726; 21727; 21728; 21729; 21730; 21731; 21732; 21733; 21734;
21735; 21736; 21737; 21738; 21739; 21740; 21741; 32212; 21742;
21743; 21744; 21745; 21746; 21747; 21748; 21749; 21750; 21751;
21752; 17790; 19154; 2810; 21753; 21754; 3894; 16099; 16100; 21755;
21756; 21757; 21758; 21759; 21760; 21761; 2821; 1974; 21762; 21763;
21764; 21765; 21766; 21767; 21768; 21769; 21770; 21771; 8866; 2823;
2824; 2825; 2826; 2827; 2828; 2829; 2830; 21772; 21773; 21774;
2831; 21775; 2832; 8889; 8890; 16108; 5468; 21776; 21777; 8896;
21778; 21779; 21780; 21781; 21782; 21783; 21784; 211; 21785; 20238;
14271; 21786; 21787; 21788; 21789; 21790; 17117; 21791; 21792;
21793; 21794; 21795; 21796; 21797; 15493; 21798; 21799; 21800;
21801; 21802; 21803; 5477; 3915; 21804; 21805; 21806; 21807; 21808;
21809; 5479; 5480; 5481; 5482; 5483; 5484; 5485; 5486; 5487; 5488;
5489; 5490; 5491; 5492; 5493; 5494; 5495; 5496; 5497; 5498; 5499;
5500; 5501; 5502; 5503; 5504; 5505; 5506; 5507; 5508; 5509; 5510;
5511; 5512; 5513; 5514; 5515; 5516; 5517; 5518; 5519; 5520; 5521;
5522; 5523; 5524; 5525; 5526; 5527; 5528; 5529; 5530; 5531; 5532;
5533; 5534; 5535; 5536; 5537; 5538; 5539; 5540; 5541; 5542; 5543;
5544; 5545; 5546; 5547; 5548; 5549; 5550; 5551; 5552; 5553; 5554;
5555; 5556; 5557; 5558; 5559; 5560; 5561; 5562; 5563; 5564; 5565;
5566; 5567; 5568; 5569; 5570; 5571; 5572; 5573; 5574; 5575; 5576;
5577; 5578; 5579; 5580; 5581; 5582; 5583; 5584; 5585; 5586; 5587;
5588; 5589; 5590; 5591; 5592; 5593; 5594; 5595; 5596; 5597; 5598;
5599; 5600; 5601; 5602; 5603; 5604; 5605; 5606; 5607; 5608; 5609;
5610; 5611; 5612; 5613; 5614; 5615; 16124; 8916; 8917; 21810;
21811; 21812; 21813; 21814; 21815; 25424; 21816; 16133; 21817;
21818; 21819; 21820; 17820; 17821; 14293; 21821; 21822; 21823;
21824; 20745; 20746; 20747; 17123; 21825; 21826; 8959; 2859; 21827;
21828; 21829; 21830; 21831; 21832; 21833; 21834; 21835; 3948;
21836; 21838; 1201; 1996; 16141; 21839; 21840; 21841; 21842; 17129;
17130; 3952; 21843; 21844; 12815; 32214; 21845; 21846; 32215;
21847; 21848; 21849; 21850; 3964; 21851; 21852; 5682; 5683; 5684;
21853; 21854; 21855; 21856; 21857; 21858; 21859; 3972; 3973; 21861;
21862; 21863; 5686; 7436; 21864; 21865; 21866; 21867; 21868; 21869;
2881; 2882; 9061; 9062; 9063; 21870; 21871; 21872; 265; 266; 21873;
21874; 14343; 14344; 14345; 3986; 21876; 21877; 21878; 21879;
21880; 21881; 2891; 16168; 19218; 19219; 21882; 21883; 19220;
19221; 19222; 19223; 21884; 21885; 19224; 19225; 19226; 19227;
21886; 7452; 7453; 7454; 7455; 9097; 21887; 21889; 16170; 16171;
21890; 21891; 21892; 21893; 21894; 21895; 21896; 9119; 21897;
21898; 21899; 21900; 21901; 21902; 21903; 9139; 21904; 5739; 21905;
17888; 19241; 19243; 19244; 19245; 4016; 4017; 4018; 7468; 21907;
21908; 4021; 19256; 21909; 16178; 9166; 9167; 14363; 2039; 19261;
9179; 9180; 9181; 9182; 9183; 21911; 16188; 21912; 21913; 21914;
21915; 21916; 21917; 9187; 9220; 21918; 21919; 21920; 21921; 14372;
21922; 21923; 21924; 21925; 19280; 21926; 21927; 4035; 1258; 2911;
2912; 32217; 5777; 21928; 7483; 7484; 7485; 21929; 21930; 1262;
1263; 1264; 1265; 1266; 1267; 9264; 9265; 9266; 9267; 9268; 9269;
9270; 13490; 21931; 21932; 25454; 21933; 2922; 4051; 4052; 21934;
21935; 21936; 21937; 21939; 21940; 21941; 1270; 21942; 5789; 4061;
21943; 19299; 1277; 21944; 21945; 9308; 20813; 21948; 327; 21949;
21950; 21951; 21952; 21953; 21954; 21955; 21956; 21957; 21958;
21959; 4075; 21960; 16229; 21961; 21962; 21963; 21964; 21965; 336;
21966; 21967; 21968; 21969; 21970; 21971; 21972; 9326; 14410;
21973; 9329; 21974; 21975; 9332; 21976; 21977; 21978; 21979; 12857;
21980; 21981; 21982; 21983; 13525; 343; 21984; 21985; 20825; 16246;
21986; 15540; 15541; 7521; 21987; 21988; 12863; 21989; 21991;
13527; 13528; 21992; 16257; 20830; 21993; 21994; 19328; 19329;
19330; 21996; 20833; 21997; 21998; 21999; 22000; 32220; 22001;
17955; 17956; 17957; 22002; 22003; 22004; 22005; 22006; 361; 12869;
12870; 22007; 17178; 16290; 22008; 22009; 22010; 22011; 22012;
9416; 9417; 22013; 22014; 22015; 22016; 22017; 22018; 22019; 22020;
22021; 22022
[0179] The following SEQ ID NOs correspond to the amino acid
sequences of male organ, prostate-specific proteins identified
using MPSS as described in Table 42A and Example 7: 22023; 22024;
16304; 22025; 22026; 22027; 22028; 4131; 4132; 4133; 4134; 22029;
13537; 9459; 22030; 2100; 1315; 9463; 14456; 12164; 12165; 12166;
22031; 22032; 22033; 22034; 9472; 9473; 22035; 22036; 22037; 22038;
22039; 22040; 22041; 22042; 22043; 22044; 22045; 2989; 2990; 2991;
22046; 22047; 22048; 383; 1323; 384; 5935; 22049; 22050; 22051;
22052; 22053; 22054; 22055; 5936; 2113; 1324; 1325; 1326; 22056;
2994; 2995; 1327; 22057; 22058; 22059; 9515; 22060; 22061; 22062;
22063; 22064; 5944; 5945; 4171; 4172; 22065; 3006; 9526; 15579;
2119; 5956; 5957; 5958; 5959; 9528; 22066; 5960; 1330; 17989;
22067; 22069; 7568; 397; 22070; 22071; 9554; 22072; 22073; 22074;
22075; 22076; 22077; 32222; 22078; 22079; 22080; 22081; 22082;
22083; 22084; 22085; 22086; 22087; 22088; 19380; 19381; 22089;
22090; 16334; 22091; 2132; 22092; 22093; 22094; 22095; 5979; 22096;
22097; 12207; 22098; 22099; 22100; 16340; 22101; 19387; 19388;
19389; 19390; 19391; 19392; 19393; 19394; 5990; 5991; 22102; 22103;
22104; 22105; 4219; 22107; 22108; 19412; 22109; 1357; 20870; 19419;
424; 22110; 22111; 22112; 22113; 9628; 22114; 22115; 22116; 22117;
14514; 14515; 22118; 19421; 19422; 32223; 22119; 22120; 22121;
20872; 22122; 22123; 12223; 22124; 14521; 22125; 22126; 9654;
22127; 2168; 2169; 19447; 19448; 19449; 22128; 22129; 22130; 22131;
7612; 15599; 16398; 22133; 15604; 22134; 22135; 9684; 18058; 22136;
7613; 22137; 22138; 7615; 22140; 22141; 458; 9694; 9695; 22142;
22143; 22144; 22145; 22146; 22147; 22148; 22149; 22150; 20885;
20886; 22151; 22152; 22153; 22154; 22155; 9736; 6054; 22156; 22157;
22158; 12238; 3055; 22159; 22160; 22161; 1384; 22162; 22163; 1386;
469; 9750; 9751; 22164; 22165; 22166; 22167; 1387; 2184; 22168;
15617; 3061; 22169; 22170; 9786; 14555; 14556; 22171; 22172; 22173;
20889; 20890; 22174; 22175; 22176; 17225; 17226; 17227; 6076; 6077;
9798; 9799; 9804; 9805; 22177; 22178; 22179; 14561; 22180; 2196;
22181; 22182; 22183; 22184; 22185; 22186; 22187; 22188; 22189;
22190; 7638; 7639; 9836; 4287; 18089; 22192; 22193; 22194; 22195;
22196; 22197; 22198; 22199; 22200; 22201; 22202; 19482; 22203;
22204; 19486; 489; 490; 491; 492; 6088; 6090; 6091; 6092; 6093;
6094; 22205; 1405; 1406; 22206; 22207; 22208; 9875; 22209; 18105;
18106; 22210; 22211; 9879; 9880; 1415; 22212; 22213; 3086; 3087;
499; 500; 13631; 22214; 22215; 22216; 22217; 22218; 22219; 22220;
22221; 22222; 22223; 22224; 22225; 22226; 7669; 505; 9924; 9925;
9928; 22227; 1425; 22228; 22229; 22230; 14602; 22232; 22233; 22234;
6135; 7676; 22235; 22236; 22237; 524; 22238; 2237; 9969; 9970;
9971; 9972; 2238; 22239; 4345; 4346; 4347; 4348; 4349; 4350; 22240;
22241; 9994; 9995; 22242; 3109; 10002; 4370; 3111; 22243; 15635;
6193; 6194; 6195; 6196; 6197; 6198; 6199; 6200; 6201; 6202; 6203;
6204; 6205; 6206; 6207; 6208; 6209; 22244; 22245; 22246; 10028;
14629; 14630; 14631; 14633; 14634; 14636; 22247; 22248; 22249;
3138; 22250; 22251; 22252; 22253; 22254; 22255; 22256; 22257;
22258; 22259; 22260; 22261; 22262; 22263; 22264; 20965; 22265;
22266; 22267; 3148; 6231; 6232; 10063; 10064; 10065; 10066; 10067;
10068; 10069; 10070; 22268; 22269; 22270; 22271; 22272; 22273;
22274; 22275; 22276; 22277; 22278; 22279; 22280; 22281; 32224;
22282; 22283; 22284; 22285; 4416; 22286; 22287; 22288; 555; 22290;
22291; 22292; 22293; 22294; 22295; 22297; 22298; 22300; 22301;
22302; 22303; 32225; 22304; 22305; 22306; 22308; 22309; 22310;
22311; 22312; 22313; 22314; 22315; 22316; 22317; 22318; 22319;
22320; 22321; 22322; 22323; 22324; 22325; 22326; 22327; 22328;
32226; 22329; 22330; 22331; 22332; 22333; 22334; 22335; 22336;
22337; 22338; 22339; 18259; 19558; 3166; 22340; 22341; 4443; 16496;
16497; 22342; 22343; 22344; 22345; 22346; 22347; 22348; 3177; 2273;
22349; 22350; 22351; 22352; 22353; 22354; 22355; 22356; 22357;
22358; 10194; 3179; 3180; 3181; 3182; 3183; 3184; 3185; 3186;
22359; 22360; 22361; 3187; 22362; 3188; 10217; 10218; 16505; 6302;
22363; 22364; 10224; 22365; 22366; 22367; 22368; 22369; 22370;
22371; 579; 22372; 20379; 14685; 22373; 22374; 22375; 22376; 22377;
17277; 22378; 22379; 22380; 22381; 22382; 22383; 22384; 15669;
22385; 22386; 22387; 22388; 22389; 22390; 6311; 4464; 22391; 22392;
22393; 22394; 22395; 22396; 6313; 6314; 6315; 6316; 6317; 6318;
6319; 6320; 6321; 6322; 6323; 6324; 6325; 6326; 6327; 6328; 6329;
6330; 6331; 6332; 6333; 6334; 6335; 6336; 6337; 6338; 6339; 6340;
6341; 6342; 6343; 6344; 6345; 6346; 6347; 6348; 6349; 6350; 6351;
6352; 6353; 6354; 6355; 6356; 6357; 6358; 6359; 6360; 6361; 6362;
6363; 6364; 6365; 6366; 6367; 6368; 6369; 6370; 6371; 6372; 6373;
6374; 6375; 6376; 6377; 6378; 6379; 6380; 6381; 6382; 6383; 6384;
6385; 6386; 6387; 6388; 6389; 6390; 6391; 6392; 6393; 6394; 6395;
6396; 6397; 6398; 6399; 6400; 6401; 6402; 6403; 6404; 6405; 6406;
6407; 6408; 6409; 6410; 6411; 6412; 6413; 6414; 6415; 6416; 6417;
6418; 6419; 6420; 6421; 6422; 6423; 6424; 6425; 6426; 6427; 6428;
6429; 6430; 6431; 6432; 6433; 6434; 6435; 6436; 6437; 6438; 6439;
6440; 6441; 6442; 6443; 6444; 6445; 6446; 6447; 6448; 6449; 16521;
10244; 10245; 22397; 22398; 22399; 22400; 22401; 22402; 25564;
22403; 16530; 22404; 22405; 22406; 22407; 18289; 18290; 14707;
22408; 22409; 22410; 22411; 21012; 21013; 21014; 17283; 22412;
22413; 10287; 3215; 22414; 22415; 22416; 22417; 22418; 22419;
22420; 22421; 22422; 4497; 22423; 22425; 1484; 2295; 16538; 22426;
22427; 22428; 22429; 17289; 17290; 4501; 22430; 22431; 13023;
32228; 22432; 22433; 32229; 22434; 22435; 22436; 22437; 4513;
22438; 22439; 6516; 6517; 6518; 22440; 22441; 22442; 22443; 22444;
22445; 22446; 4521; 4522; 22448; 22449; 22450; 6520; 7766; 22451;
22452; 22453; 22454; 22455; 22456; 3237; 3238; 10389; 10390; 10391;
22457; 22458; 22459; 633; 634; 22460; 22461; 14757; 14758; 14759;
4535; 22463; 22464; 22465; 22466; 22467; 22468; 3247; 16565; 19622;
19623; 22469; 22470; 19624; 19625; 19626; 19627; 22471; 22472;
19628; 19629; 19630; 19631; 22473; 7782; 7783; 7784; 7785; 10425;
22474; 22476; 16567; 16568; 22477; 22478; 22479; 22480; 22481;
22482; 22483; 10447; 22484; 22485; 22486; 22487; 22488; 22489;
22490; 10467; 22491; 6573; 22492; 18357; 19645; 19647; 19648;
19649; 4565; 4566; 4567; 7798; 22494; 22495; 4570; 19660; 22496;
16575; 10494; 10495; 14777; 2338; 19665; 10507; 10508; 10509;
10510; 10511; 22498; 16585; 22499; 22500; 22501; 22502; 22503;
22504; 10515; 10548; 22505; 22506; 22507; 22508; 14786; 22509;
22510; 22511; 22512; 19684; 22513; 22514; 4584; 1541; 3267; 3268;
32231; 6611; 22515; 7813; 7814; 7815; 22516; 22517; 1545; 1546;
1547; 1548; 1549; 1550; 10592; 10593; 10594; 10595; 10596; 10597;
10598; 13757; 22518; 22519; 25594; 22520; 3278; 4600; 4601; 22521;
22522; 22523; 22524; 22526; 22527; 22528; 1553; 22529; 6623; 4610;
22530; 19703; 1560; 22531; 22532; 10636; 21080; 22535; 695; 22536;
22537; 22538; 22539; 22540; 22541; 22542; 22543; 22544; 22545;
22546; 4624; 22547; 16626; 22548; 22549; 22550; 22551; 22552; 704;
22553; 22554; 22555; 22556; 22557; 22558; 22559; 10654; 14824;
22560; 10657; 22561; 22562; 10660; 22563; 22564; 22565; 22566;
13065; 22567; 22568; 22569; 22570; 13792; 711; 22571; 22572; 21092;
16643; 22573; 15716; 15717; 7851; 22574; 22575; 13071; 22576;
22578; 13794; 13795; 22579; 16654; 21097; 22580; 22581; 19732;
19733; 19734; 22583; 21100; 22584; 22585; 22586; 22587; 32234;
22588; 18424; 18425; 18426; 22589; 22590; 22591; 22592; 22593; 729;
13077; 13078; 22594; 17338; 16687; 22595; 22596; 22597; 22598;
22599; 10744; 10745; 22600; 22601; 22602; 22603; 22604; 22605;
22606; 22607; 22608; 22609.
[0180] The following SEQ ID NOs correspond to the amino acid
sequences of adrenal gland-specific proteins identified using MPSS
and that have been identified by mass spectrometry as described in
Table 43A: 370; 371; 373; 374; 375; 380; 381; 383; 384; 386; 387;
388; 392; 393; 394; 397; 398; 399; 400; 401; 404; 406; 407; 408;
409; 410; 411; 412; 421; 423; 424; 426; 427; 428; 429; 430; 431;
432; 440; 441; 442; 443; 444; 449; 450; 451; 454; 456; 459; 460;
466; 467; 468; 469; 470; 471; 479; 480; 485; 488; 497; 499; 500;
503; 507; 511; 516; 517; 518; 519; 522; 523; 524; 525; 526; 527;
528; 532; 538; 541; 543; 544; 546; 547; 553; 556; 559; 563; 564;
565; 566; 567; 568; 569; 570; 572; 574; 576; 577; 590; 598; 608;
609; 611; 612; 613; 614; 615; 623; 624; 625; 628; 629; 630; 636;
637; 638; 639; 641; 642; 643; 644; 645; 648; 649; 653; 656; 659;
660; 661; 662; 663; 668; 670; 673; 674; 675; 676; 678; 686; 687;
688; 689; 699; 707; 708; 709; 710; 712; 713; 714; 715; 716; 717;
723; 724; 725; 730; 731.
[0181] The following SEQ ID NOs correspond to the amino acid
sequences of bladder-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 1313; 1316; 1317; 1318; 1319; 1322; 1323; 1324; 1325;
1326; 1327; 1330; 1333; 1341; 1344; 1346; 1347; 1348; 1349; 1350;
1351; 1353; 1354; 1355; 1357; 1358; 1359; 1364; 1369; 1372; 1373;
1374; 1378; 1379; 1380; 1382; 1383; 1384; 1385; 1386; 1388; 1389;
1390; 1392; 1393; 1394; 1397; 1400; 1401; 1402; 1404; 1415; 1417;
1418; 499; 1419; 1420; 1425; 1428; 517; 1430; 1432; 1433; 1434;
1442; 1443; 1447; 1453; 1454; 1469; 1470; 1471; 1473; 1474; 1475;
1476; 1477; 1481; 1482; 1484; 1485; 1487; 1488; 1490; 1491; 1495;
1499; 1500; 1502; 1505; 1506; 1507; 1511; 1516; 1518; 1519; 1520;
1526; 1527; 1529; 1530; 1531; 1532; 1536; 1537; 1540; 1555; 1556;
1557; 1561; 1564; 1565; 1568; 1569; 1570; 1571; 1572; 1574; 1575;
1576; 1577; 1578; 1581; 1582; 1583; 1585; 1588; 1590; 1591; 1592;
1594.
[0182] The following SEQ ID NOs correspond to the amino acid
sequences of bone marrow-specific proteins identified using MPSS
and that have been identified by mass spectrometry as described in
Table 43A: 2095; 2096; 2097; 2098; 2099; 2100; 2104; 2108; 2109;
2110; 2111; 2112; 2113; 2118; 2119; 2120; 2125; 2136; 2138; 2143;
2144; 2145; 2148; 2149; 2151; 2152; 2153; 2155; 2160; 2167; 2171;
2172; 2176; 2177; 2178; 2179; 470; 2183; 2184; 2185; 2191; 2192;
2195; 2218; 2219; 2220; 2222; 2224; 2227; 2228; 2230; 2231; 2232;
2233; 2234; 2235; 2242; 2243; 2244; 2245; 2246; 2250; 2252; 2253;
2256; 2257; 2258; 2259; 2260; 2261; 2262; 2263; 2265; 2266; 2267;
2268; 2269; 2271; 2274; 2283; 2290; 2292; 2293; 1484; 2296; 2297;
2306; 2308; 2309; 2310; 2311; 2312; 2316; 2317; 2318; 2319; 2320;
2321; 2322; 1511; 2326; 2328; 2330; 2336; 2337; 2338; 2339; 2340;
2341; 2342; 2343; 2344; 2345; 2346; 2347; 2348; 2349; 2350; 2353;
2354; 2356; 2357; 2358; 2363; 2372; 2373; 2375; 2380; 2381; 2382;
2385; 2386; 2387; 2388; 2389; 2393.
[0183] The following SEQ ID NOs correspond to the amino acid
sequences of brain amygdala-specific proteins identified using MPSS
and that have been identified by mass spectrometry as described in
Table 43A: 2980; 2098; 2099; 2982; 2983; 2984; 2985; 2986; 2987;
2988; 2989; 2993; 2998; 3001; 3003; 3004; 3005; 3006; 3007; 1333;
3017; 3019; 3020; 408; 3023; 3024; 3025; 3026; 3033; 3035; 3036;
1358; 3037; 3040; 3041; 3042; 3043; 440; 441; 442; 443; 444; 3050;
3051; 3052; 3055; 3056; 3057; 3058; 3059; 3060; 3071; 3079; 3080;
3082; 3083; 2219; 2222; 3091; 3092; 3093; 3095; 3097; 3098; 3099;
522; 3101; 3110; 3112; 3139; 3141; 3142; 3143; 3148; 3164; 3166;
3167; 3169; 3171; 3172; 3177; 3187; 3189; 3190; 3191; 1470; 1471;
3198; 3199; 1481; 3200; 3201; 3202; 3203; 3204; 3218; 612; 613;
3220; 3221; 3222; 3223; 3227; 3228; 3229; 3230; 3231; 3232; 3233;
628; 3234; 3235; 3237; 3238; 3239; 3240; 3241; 3242; 3245; 3247;
3248; 3255; 1520; 3256; 3257; 3258; 3259; 3261; 3262; 3263; 3267;
3268; 3270; 3272; 3273; 3275; 3276; 3277; 3278; 3279; 3280; 3282;
3283; 3284; 3285; 3292; 3294; 3295; 3297; 3299; 3301; 3307; 3308;
3309; 3310; 3311; 3312; 714; 3314; 3315; 3316; 3317; 3324; 2386;
3327; 3328; 2393.
[0184] The following SEQ ID NOs correspond to the amino acid
sequences of Brain Caudate Nucleus-specific proteins identified
using MPSS and that have been identified by mass spectrometry as
described in Table 43A: 4135; 4138; 2983; 2984; 2985; 4139; 4141;
4142; 4143; 4147; 4148; 4151; 4152; 4153; 388; 4158; 4163; 4165;
4168; 4171; 4172; 1330; 4177; 4178; 4179; 4190; 4194; 4205; 4206;
4207; 4208; 3026; 410; 4211; 4213; 4215; 4217; 4218; 4219; 4220;
4221; 4223; 4224; 4226; 4227; 4229; 426; 427; 428; 4238; 1364;
4239; 4240; 4244; 4251; 4252; 4253; 4254; 4255; 4258; 4263; 4264;
4266; 4269; 4270; 1386; 4274; 4278; 4280; 4281; 4282; 4283; 4284;
4288; 4290; 4295; 4296; 4297; 4304; 4305; 4312; 4313; 4314; 4316;
4319; 4323; 4324; 4329; 4333; 4336; 4339; 4341; 4344; 4345; 4346;
4347; 4348; 4349; 4350; 4356; 4371; 3112; 4372; 4374; 4375; 4407;
4408; 4409; 4410; 4425; 4432; 4437; 4439; 4444; 4445; 4446; 4455;
4459; 4462; 4463; 4466; 4467; 4468; 4469; 4470; 4471; 4475; 4478;
4479; 4481; 4483; 4484; 4487; 4496; 4498; 612; 613; 4500; 4503;
4504; 4506; 4510; 4511; 4512; 4513; 4517; 4518; 4521; 4525; 4526;
4535; 4541; 4542; 4544; 4552; 4553; 4554; 4559; 4560; 3255; 4563;
4564; 4565; 4566; 4567; 4570; 4572; 4574; 4576; 4577; 3259; 4580;
4586; 4587; 4588; 4590; 673; 674; 675; 4594; 4595; 4596; 4597;
4598; 4599; 4604; 4605; 4608; 4609; 4611; 4612; 4617; 3284; 3285;
4628; 4630; 4632; 4635; 1568; 1569; 1570; 1571; 1572; 1574; 4638;
4640; 4641; 4642; 4643; 4644; 4645; 4649; 4654; 4655; 4660; 4674;
4677; 2393.
[0185] The following SEQ ID NOs correspond to the amino acid
sequences of Brain Cerebellum-specific proteins identified using
MPSS and that have been identified by mass spectrometry as
described in Table 43A: 5904; 2095; 2096; 2097; 5906; 5907; 5908;
5909; 5910; 5911; 5912; 2098; 2099; 5913; 5914; 5915; 5916; 5917;
5918; 5919; 5920; 5921; 5922; 5923; 5924; 5925; 5930; 5931; 5932;
5933; 5935; 2998; 5939; 5940; 5941; 5942; 5943; 5944; 5946; 3006;
5961; 5974; 5976; 5982; 5983; 5987; 5988; 5989; 410; 2138; 5997;
6000; 6001; 6011; 6012; 426; 427; 428; 6015; 6019; 6021; 6024;
6025; 6026; 6029; 6030; 4251; 456; 6037; 6038; 6042; 4255; 6049;
6052; 6054; 6056; 6057; 6058; 6059; 6060; 6061; 6064; 6065; 6066;
469; 6071; 6074; 6076; 6079; 6080; 6088; 6090; 6091; 6092; 6093;
6094; 3082; 3083; 6101; 6102; 2219; 6104; 6108; 6109; 6110; 6111;
6112; 6113; 6114; 6116; 6117; 6118; 6120; 6121; 6126; 6128; 6129;
6130; 6131; 6132; 4329; 6134; 6135; 6136; 6137; 6138; 6142; 1434;
6156; 6157; 4371; 6171; 6173; 6174; 6180; 6181; 6182; 6183; 6189;
6190; 6191; 6210; 6211; 6212; 6224; 6225; 6226; 6231; 6233; 6273;
6284; 6292; 6293; 6296; 6297; 6298; 6303; 6306; 6307; 1473; 1474;
1475; 1476; 1477; 6311; 4466; 6450; 6451; 6453; 6454; 6455; 6456;
6457; 6458; 6459; 6460; 3200; 6461; 6462; 6465; 3204; 6466; 6467;
6470; 6472; 6473; 6475; 6476; 1485; 6478; 6479; 6480; 6482; 6483;
6484; 6485; 6487; 6488; 6489; 6490; 6492; 6493; 6494; 6496; 6500;
1495; 6501; 6506; 4510; 6508; 3229; 3230; 3231; 6512; 6514; 3232;
6515; 1499; 6519; 6524; 6528; 6532; 6533; 6538; 6541; 6542; 6544;
6545; 6546; 6547; 6548; 6549; 6550; 6551; 6552; 6556; 6557; 6558;
6559; 6563; 6564; 6579; 6581; 6585; 6588; 6589; 6593; 1532; 1536;
1537; 6599; 6610; 6611; 6613; 3273; 3275; 6619; 6620; 4605; 6622;
6623; 6624; 6626; 6628; 6631; 6633; 6636; 6638; 4628; 6639; 6643;
6644; 3308; 3309; 3310; 3311; 3312; 1577; 6658; 3314; 3315; 6663;
6665; 6666; 6668; 6672; 6674; 6675; 2387; 2388; 2389; 6725; 6732;
6735; 4677; 2393.
[0186] The following SEQ ID NOs correspond to the amino acid
sequences of Brain Corpus Callosum-specific proteins identified
using MPSS and that have been identified by mass spectrometry as
described in Table 43A: 7543; 7544; 7545; 7547; 7548; 7552; 7553;
2989; 7558; 2113; 7560; 7561; 7562; 7563; 7568; 4178; 7572; 7591;
7592; 7593; 7598; 7599; 7601; 4229; 7603; 7604; 7605; 3042; 3043;
7611; 7613; 7614; 7615; 7617; 7618; 3055; 3056; 3057; 3058; 6056;
6057; 6058; 6059; 6060; 6061; 7624; 7625; 7627; 7628; 7648; 4295;
4296; 7658; 7662; 7663; 7664; 7666; 7667; 3091; 3092; 7668; 7669;
7671; 7672; 6126; 2234; 2235; 7674; 7675; 6135; 7676; 7677; 7678;
7679; 7680; 7681; 7686; 7690; 7691; 7697; 7714; 7716; 559; 4446;
3177; 7726; 7729; 7733; 7734; 4475; 7736; 7737; 7742; 4487; 7756;
7757; 7760; 7761; 7762; 7763; 7767; 7768; 7770; 7776; 7777; 7778;
7779; 7780; 7782; 7783; 7784; 7785; 7786; 7791; 7792; 7793; 7795;
7796; 7797; 7798; 7801; 7802; 7803; 7804; 7805; 7813; 7814; 7836;
7838; 7840; 7841; 7844; 7846; 7847; 7851; 7852; 7853; 7861; 7863;
3327; 3328; 7867; 7869; 7871.
[0187] The following SEQ ID NOs correspond to the amino acid
sequences of Brain Fetal-specific proteins identified using MPSS
and that have been identified by mass spectrometry as described in
Table 43A: 5904; 2095; 2096; 2097; 9442; 9443; 9446; 9449; 9450;
9451; 9452; 9453; 9454; 9455; 9456; 9457; 9458; 9459; 2100; 9460;
9463; 9464; 9465; 9466; 9467; 9468; 9469; 9472; 9473; 9477; 2983;
2984; 2985; 9485; 9489; 2989; 9490; 9495; 9497; 5935; 4147; 9500;
9503; 2112; 9512; 2998; 9515; 3001; 7561; 7562; 9516; 9517; 5944;
9519; 9520; 9521; 9522; 9523; 4171; 4172; 3006; 9526; 9527; 9528;
9529; 9530; 9536; 9545; 9546; 9550; 9551; 4178; 7572; 9561; 9562;
9563; 9565; 9569; 9574; 3019; 3020; 9581; 4206; 9585; 9589; 1346;
1347; 1348; 1349; 1350; 410; 4217; 4218; 412; 9602; 9603; 9608;
9609; 9611; 9613; 9618; 421; 9623; 9624; 3037; 3040; 9632; 9633;
9634; 9637; 9640; 6015; 9643; 9644; 9647; 9648; 9651; 6021; 9659;
9660; 9661; 9662; 9665; 440; 441; 442; 443; 444; 9667; 9679; 9680;
9682; 9686; 9687; 9689; 4254; 9692; 9694; 9695; 9696; 9699; 9702;
9703; 3050; 3051; 3052; 9705; 9707; 9708; 9711; 9712; 9713; 9714;
9715; 9716; 9717; 9732; 9734; 9735; 9736; 6056; 6057; 6058; 6059;
6060; 6061; 9740; 9741; 9742; 9744; 9746; 9747; 469; 9750; 9751;
9753; 9754; 9755; 9762; 9763; 9769; 9770; 2184; 9771; 9772; 9773;
3060; 9776; 9777; 9778; 9779; 9780; 9781; 9784; 1388; 1389; 9785;
9786; 9787; 9788; 9789; 9790; 9791; 1392; 9795; 4280; 4281; 4282;
9797; 9798; 9801; 4283; 9804; 9808; 9809; 9823; 9824; 9828; 9833;
9844; 9845; 9853; 9864; 9868; 9870; 4295; 4296; 9875; 9876; 3080;
9877; 9883; 497; 9885; 9886; 499; 9888; 9889; 9890; 4312; 4313;
9893; 9894; 9896; 9897; 9898; 9899; 9908; 9909; 9910; 9911; 9914;
9915; 9916; 9917; 9919; 2224; 7669; 9923; 9924; 9925; 9929; 9931;
6120; 9935; 9938; 9939; 9940; 9941; 9942; 9943; 9944; 9945; 9946;
4329; 9947; 9952; 9954; 9955; 9959; 9961; 9962; 9967; 9968; 9976;
9977; 6142; 9980; 9981; 9982; 9984; 9985; 532; 9991; 9992; 9999;
10003; 10004; 10005; 10008; 10009; 10019; 10020; 543; 10027; 10029;
10030; 10031; 10032; 10034; 10035; 10036; 10039; 10042; 10044;
10046; 10051; 7690; 10055; 10056; 4407; 4408; 10082; 10161; 10162;
10164; 10167; 10170; 10173; 10174; 4444; 4445; 563; 564; 565; 566;
567; 568; 569; 570; 10176; 10178; 3172; 10186; 3177; 10196; 10199;
10200; 10201; 10204; 10205; 10213; 10219; 10220; 10224; 10225;
4459; 3190; 10237; 10238; 6311; 10239; 10240; 10244; 10245; 3200;
10248; 6462; 4475; 10254; 10255; 10256; 10258; 10259; 10260; 10261;
10264; 10267; 10268; 10269; 10273; 6470; 6472; 6473; 10277; 10278;
10279; 10280; 10281; 10285; 10286; 10287; 10289; 10290; 10294;
4487; 10295; 10296; 10298; 10299; 10301; 10316; 10324; 10327;
10331; 10333; 10336; 6500; 3227; 3228; 10338; 1495; 10347; 10348;
10349; 10350; 10351; 10352; 10353; 10354; 10355; 6514; 10358;
10360; 10362; 10364; 10366; 10371; 10372; 10373; 10375; 10376;
10383; 10384; 10385; 10386; 6528; 10388; 10389; 10390; 10391;
10392; 10393; 10394; 10398; 3242; 10399; 10400; 10401; 10403;
10404; 10407; 10408; 10409; 10410; 10411; 10413; 10414; 10415;
10416; 10423; 10424; 10425; 10426; 4541; 10437; 1511; 643; 10441;
10444; 2326; 10445; 10447; 2328; 10450; 10457; 10458; 10459; 10460;
10461; 10462; 10463; 10464; 10465; 10467; 3255; 10476; 1519; 10477;
10478; 10479; 10481; 10482; 7795; 10484; 10485; 10486; 10487;
10488; 6579; 10489; 10490; 10491; 10492; 10496; 10497; 10498;
10499; 4574; 10501; 10502; 10506; 10508; 10509; 10511; 10512;
10513; 10514; 4576; 7803; 10515; 10517; 10519; 3257; 10549; 10552;
10553; 7804; 10554; 3259; 10558; 10559; 10560; 10564; 10565; 10570;
10578; 10582; 6611; 10583; 10584; 10590; 10591; 10595; 10596;
10598; 673; 674; 675; 10599; 676; 10600; 10603; 10604; 10605;
10606; 10607; 10608; 10610; 10611; 3275; 10614; 3276; 3277; 10615;
7836; 10616; 3279; 4608; 4609; 10620; 10626; 3283; 10628; 10636;
10639; 10640; 2372; 10644; 10645; 10646; 10647; 10649; 2375; 10652;
10656; 10657; 1568; 1569; 1570; 1571; 1572; 1574; 10660; 10670;
10671; 713; 4641; 10672; 10673; 10676; 10677; 10681; 2380; 10682;
10698; 10699; 10700; 10701; 10702; 10704; 10706; 10710; 10719;
10723; 10724; 10725; 2387; 2388; 2389; 10735; 10744; 10745;
7867.
[0188] The following SEQ ID NOs correspond to the amino acid
sequences of Brain Hypothalamus-specific proteins identified using
MPSS and that have been identified by mass spectrometry as
described in Table 43A: 2980; 12162; 373; 374; 5911; 5912; 9472;
9473; 9489; 12175; 12176; 5935; 1324; 1325; 1326; 12177; 12183;
12184; 12187; 12188; 12189; 12190; 12191; 12192; 12193; 12195;
12198; 12202; 12212; 12218; 12220; 9632; 9633; 1359; 12221; 12222;
12223; 12227; 12230; 12232; 12233; 9736; 467; 6065; 6066; 12243;
12244; 12245; 7662; 12249; 7664; 7666; 7667; 7668; 12250; 12251;
12253; 12254; 12258; 12263; 12264; 6134; 522; 12267; 12268; 10004;
10019; 10020; 12276; 12277; 12279; 12280; 12281; 12282; 7691; 546;
4409; 12303; 2271; 12306; 10213; 10220; 3190; 3191; 6311; 2283;
12315; 12316; 6450; 6451; 12317; 12318; 12320; 12321; 12322; 10267;
10268; 10269; 12323; 6470; 12324; 12325; 12330; 10298; 12332;
12334; 12335; 4500; 12338; 12339; 6496; 6508; 10348; 10349; 10350;
10351; 10352; 10353; 10354; 10355; 6514; 12342; 12345; 12350;
12356; 6563; 6564; 12357; 12362; 12363; 10506; 12365; 12370; 12376;
12383; 12386; 4609; 12387; 12390; 7838; 12395; 12397; 12399; 12403;
12405; 12406; 7863; 12411; 731.
[0189] The following SEQ ID NOs correspond to the amino acid
sequences of Brain Thalamus-specific proteins identified using MPSS
and that have been identified by mass spectrometry as described in
Table 43A: 12878; 12879; 12880; 12890; 4177; 12891; 12893; 12895;
12896; 12897; 12898; 12902; 12903; 12904; 12908; 12914; 12915;
12916; 412; 12921; 12922; 12923; 12927; 12931; 12938; 12939; 4278;
12244; 12245; 12946; 12948; 9875; 12951; 12952; 9908; 7671; 7672;
4329; 12957; 12958; 12959; 1432; 12960; 12964; 6212; 12969; 10161;
13004; 13005; 13007; 13013; 13014; 13015; 6472; 6473; 13016; 13017;
13019; 13020; 10383; 10384; 10385; 13031; 7785; 13033; 13034; 4542;
6550; 6551; 13038; 13039; 13047; 13048; 13049; 676; 3275; 13051;
6622; 10639; 13053; 13054; 13055; 6644; 13062; 13063; 13064; 13065;
13069; 13071; 13073; 13076; 10735; 6725.
[0190] The following SEQ ID NOs correspond to the amino acid
sequences of Colon-specific proteins identified using MPSS and that
have been identified by mass spectrometry as described in Table
43A: 13533; 13534; 13535; 13536; 13537; 9459; 13540; 13541; 13543;
12890; 13544; 13545; 404; 2136; 13577; 13580; 13583; 13584; 13588;
9609; 2151; 2152; 13591; 13592; 13593; 13596; 13599; 13601; 13605;
469; 471; 9786; 13608; 13609; 13611; 13612; 13622; 4295; 4296;
13624; 13625; 13628; 13629; 13630; 13631; 13641; 9938; 6135; 7676;
13643; 9967; 13646; 13647; 13648; 13649; 13653; 12280; 13654;
13655; 13679; 10219; 13681; 13682; 13685; 13686; 13691; 13692;
13693; 13694; 13695; 13696; 13709; 623; 13717; 10383; 10384; 10385;
13736; 10477; 13739; 13740; 4566; 4567; 6585; 13741; 13742; 13743;
13744; 13747; 10565; 13750; 3262; 4588; 4590; 13754; 13755; 4594;
4595; 4596; 4597; 4598; 4599; 13762; 13763; 13051; 13764; 13765;
13766; 13767; 13768; 13769; 13770; 686; 13774; 13775; 13776; 13779;
13780; 13781; 13062; 13063; 13064; 13782; 13783; 13784; 13785;
13786; 13787; 7847; 13791; 13792; 13793; 13794; 13795; 730.
[0191] The following SEQ ID NOs correspond to the amino acid
sequences of heart-specific proteins identified using MPSS and that
have been identified by mass spectrometry as described in Table
43A: 5904; 14450; 14453; 1313; 14454; 9460; 14455; 14458; 14459;
14460; 14461; 14466; 383; 14469; 14470; 14471; 9497; 4163; 4165;
14474; 14475; 14478; 14479; 14480; 14481; 12193; 14488; 14489;
1344; 14492; 14494; 14495; 14496; 14500; 14501; 14502; 14503;
14504; 14505; 14508; 14511; 14512; 12927; 14516; 14517; 14523;
14524; 2178; 459; 14534; 14540; 14541; 7617; 14542; 14543; 7618;
9712; 9735; 466; 9750; 9751; 14549; 14552; 7624; 7625; 14555;
14556; 9798; 14561; 14566; 14567; 14568; 14569; 3079; 9877; 14582;
14583; 4324; 14603; 14604; 14605; 14606; 14607; 14608; 14609;
14610; 14611; 14612; 14613; 14615; 14616; 14617; 6136; 6137; 14619;
13646; 13647; 13648; 13649; 14620; 14621; 14622; 9991; 14624;
14625; 14626; 14627; 14628; 14638; 14640; 7691; 14644; 10162;
14664; 4444; 4445; 14665; 14668; 14669; 14670; 14671; 14672; 14673;
14674; 10200; 10201; 10204; 10205; 6306; 14681; 14684; 14685; 3190;
14691; 14693; 6311; 13692; 3198; 3199; 14698; 14699; 14700; 3200;
14702; 4475; 14704; 4478; 14707; 14709; 14710; 14714; 4487; 14715;
14717; 14718; 14721; 14722; 14723; 14724; 14725; 2296; 2297; 10327;
14730; 14735; 14736; 14737; 14739; 14742; 14744; 14745; 12345;
14747; 14756; 14757; 14758; 10410; 14762; 14764; 14770; 14772;
14777; 10499; 14778; 14780; 14782; 14783; 14784; 10564; 14787;
2353; 14790; 14791; 14793; 14795; 14796; 12383; 10595; 10596;
10598; 14801; 14804; 14805; 14807; 14809; 1557; 6626; 6628; 14819;
14820; 14821; 10657; 14831; 1568; 1569; 1570; 1571; 1572; 1574;
14835; 13065; 14843; 3308; 3309; 3310; 3311; 3312; 14844; 2381;
13073; 4655; 10704; 6665; 6666; 14852; 14855; 14856; 14862.
[0192] The following SEQ ID NOs correspond to the amino acid
sequences of kidney-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 15552; 1319; 15561; 15562; 15563; 15564; 2983; 2984;
15568; 15569; 15571; 15572; 15577; 15578; 15579; 4179; 15586;
15587; 4207; 15593; 15597; 15598; 15604; 15605; 2176; 15607; 15612;
15613; 15614; 15616; 3059; 15617; 15620; 1415; 15627; 15628; 15629;
15630; 15631; 15634; 15635; 15645; 15657; 14674; 15658; 15659;
7742; 15674; 15675; 15676; 15677; 15678; 15681; 15683; 15690;
15691; 15692; 15693; 15695; 2337; 10477; 4577; 10549; 15696; 3263;
14793; 15707; 15708; 15709; 12390; 3292; 15711; 15712; 15713;
15714; 15718; 15719; 15720; 15721; 15726.
[0193] The following SEQ ID NOs correspond to the amino acid
sequences of lung-specific proteins identified using MPSS and that
have been identified by mass spectrometry as described in Table
43A: 16302; 16303; 16304; 5911; 5912; 1318; 16305; 16306; 2986;
2987; 2988; 16310; 16311; 16312; 16313; 16314; 5940; 3001; 3006;
16318; 9527; 16319; 16320; 16321; 5961; 16322; 16324; 16327; 16333;
409; 1346; 1347; 1348; 1349; 1350; 12914; 16341; 16342; 16343;
16344; 16345; 16346; 16347; 16348; 16349; 16350; 16351; 16352;
16353; 16354; 16355; 16356; 16357; 16358; 2143; 16378; 16379;
16382; 16383; 9623; 15593; 16384; 16385; 16389; 16390; 16391;
16392; 16395; 16399; 16401; 3055; 3056; 3057; 3058; 16403; 1386;
14549; 16405; 1394; 16412; 16413; 14566; 14567; 14568; 14569;
16428; 16429; 16431; 9883; 9899; 6111; 6112; 6113; 6114; 16437;
16440; 14610; 511; 16442; 16443; 16444; 16445; 16446; 16447; 522;
12267; 6136; 6137; 16448; 16451; 16452; 527; 9981; 9982; 4345;
4346; 4347; 4348; 4349; 4350; 16455; 6225; 6226; 16458; 16459;
2246; 16464; 16495; 16498; 16500; 16504; 16505; 16507; 16508;
16511; 16512; 2283; 16522; 16530; 16532; 16533; 16535; 16536; 1484;
16540; 16541; 4517; 16548; 16549; 16558; 16559; 16560; 16561;
16563; 16564; 16565; 16567; 16568; 4552; 4553; 4554; 16571; 10484;
10485; 16574; 16578; 16580; 16581; 16582; 16583; 1527; 16585;
16586; 1529; 2342; 2343; 2344; 2345; 16587; 16593; 16597; 16602;
16603; 16604; 16605; 16610; 16620; 14819; 16621; 16622; 10639;
15711; 16626; 16627; 3299; 16631; 16634; 1569; 1571; 1572; 1574;
16644; 16645; 2381; 16646; 16647; 16650; 16651; 16655; 16658;
16659; 16662; 16664; 6663; 16665; 16697.
[0194] The following SEQ ID NOs correspond to the amino acid
sequences of mammary gland-specific proteins identified using MPSS
and that have been identified by mass spectrometry as described in
Table 43A: 17184; 17185; 17186; 17187; 17188; 7558; 17189; 17190;
17191; 5935; 5941; 5942; 17196; 17197; 17198; 17200; 17201; 408;
3025; 17209; 17210; 17211; 17212; 17213; 17214; 17218; 17219;
17220; 3055; 3056; 3057; 3058; 9786; 17228; 6088; 6090; 6091; 6092;
6093; 6094; 499; 12250; 12251; 17243; 518; 519; 17246; 532; 17258;
17259; 17261; 17262; 17263; 3171; 17275; 3191; 17281; 17283; 17284;
10298; 17288; 17292; 17293; 17298; 17301; 17302; 17303; 17305;
17306; 17307; 1519; 13740; 10486; 16574; 17312; 17313; 17314;
17315; 2342; 2343; 2344; 2345; 16587; 6610; 13051; 16622; 14821;
17326; 1564; 17329; 17332; 14835; 17333; 7846; 13065; 17335; 1582;
6672; 17336; 723.
[0195] The following SEQ ID NOs correspond to the amino acid
sequences of monocyte-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 9459; 2983; 2984; 2985; 17975; 17976; 17977; 17978;
12175; 12176; 17979; 17980; 17981; 1327; 12177; 17983; 17986; 392;
393; 17994; 17995; 17996; 17998; 18001; 18007; 14495; 14496; 18020;
18026; 18027; 1354; 1355; 14500; 14501; 14502; 14503; 14504; 18032;
9624; 3040; 16384; 16385; 18050; 9659; 9660; 18051; 18052; 18053;
18054; 440; 441; 442; 443; 444; 18058; 18059; 7614; 15607; 9699;
18063; 18064; 18065; 18066; 16401; 9735; 3055; 3056; 3057; 3058;
18071; 18072; 18073; 18074; 18075; 18076; 18077; 18078; 18079;
9787; 18081; 18083; 4280; 4281; 4282; 12244; 12245; 18088; 18092;
18093; 18094; 6088; 6090; 6091; 6092; 6093; 6094; 18102; 18105;
18106; 18111; 497; 1417; 18114; 500; 18115; 18119; 18121; 18122;
18123; 18126; 18127; 18129; 18130; 18132; 18133; 18134; 18136; 511;
14616; 14617; 18146; 18147; 18148; 18149; 18150; 18151; 18153;
18154; 18155; 18156; 18157; 6156; 15635; 18166; 2246; 18175; 6231;
18258; 18262; 18264; 13007; 18267; 3191; 18281; 3198; 3199; 18284;
18286; 18287; 14704; 10258; 18289; 18291; 18292; 18293; 18297;
18309; 18310; 18311; 10327; 18314; 16540; 1490; 1491; 18317; 18318;
18319; 18322; 2306; 18327; 18328; 18329; 18330; 3235; 4525; 4526;
6532; 18332; 18333; 2316; 16565; 18335; 18336; 18337; 10441; 18342;
18345; 2328; 18354; 18355; 18358; 10491; 10498; 1526; 18364; 18366;
2342; 2343; 2344; 2345; 18371; 18373; 18374; 10583; 18377; 18378;
3276; 3277; 10616; 18392; 18393; 18394; 10649; 18396; 18401; 18403;
18404; 18405; 18406; 14844; 10681; 18410; 3316; 3317; 18424;
18425.
[0196] The following SEQ ID NOs correspond to the amino acid
sequences of pancreas-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 18873; 18874; 18875; 18876; 17978; 18881; 18895;
18901.
[0197] The following SEQ ID NOs correspond to the amino acid
sequences of PBL-specific proteins identified using MPSS and that
have been identified by mass spectrometry as described in Table
43A: 19351; 16302; 19353; 14455; 19357; 4138; 4139; 4141; 4142;
9485; 19358; 2993; 9497; 19360; 4148; 19362; 19363; 9528; 4177;
19369; 19371; 19373; 19374; 19375; 19376; 19377; 4178; 19379;
19380; 19381; 1344; 410; 19397; 19398; 14500; 14501; 14502; 14503;
14504; 4224; 19415; 19418; 19419; 19423; 19433; 19435; 19438;
19439; 19440; 19441; 19442; 19443; 19444; 19445; 19452; 19453;
19454; 1369; 19455; 6026; 6030; 1378; 19458; 19460; 18073; 18074;
18075; 18076; 18077; 18078; 18079; 6101; 19495; 9883; 19500; 7664;
7666; 7667; 3091; 3092; 1419; 1420; 18119; 15627; 19509; 19510;
7671; 7672; 16444; 19512; 18146; 19516; 6142; 13646; 13647; 13648;
13649; 12958; 12959; 9981; 9982; 19518; 19522; 19523; 19524; 19533;
7690; 19535; 18175; 19565; 19566; 12306; 19570; 19571; 10237; 1470;
1471; 19572; 19575; 18287; 590; 18291; 19578; 19579; 19588; 19589;
19590; 19591; 19592; 19593; 19594; 19595; 19596; 19601; 19603;
19604; 19605; 19606; 19608; 10338; 10360; 19611; 19612; 18330;
13031; 636; 637; 19618; 19620; 19633; 19634; 19635; 19636; 16567;
16568; 644; 645; 2337; 19644; 19645; 13740; 19646; 19647; 19648;
19649; 19650; 19651; 10487; 10488; 19653; 19654; 19655; 19656;
19657; 19658; 16574; 19659; 19660; 19661; 19663; 19664; 19665;
19668; 1527; 19673; 19674; 2341; 19675; 19676; 1530; 19678; 19682;
12370; 19683; 19684; 6611; 19688; 19692; 10616; 19695; 3279; 19702;
19707; 10645; 10646; 13776; 19710; 4635; 19719; 19720; 1582; 2380;
10704; 19730; 19736; 19738; 19740; 19742; 19745.
[0198] The following SEQ ID NOs correspond to the amino acid
sequences of pituitary gland-specific proteins identified using
MPSS and that have been identified by mass spectrometry as
described in Table 43A: 5906; 5907; 5908; 5909; 5910; 5911; 5912;
20276; 20280; 20281; 20282; 20283; 7561; 7562; 20293; 6000; 6001;
20306; 20307; 20308; 20309; 6011; 20311; 20312; 20314; 20315;
20319; 20320; 20321; 20322; 1386; 20323; 13608; 13609; 20327;
20333; 1418; 20350; 499; 6134; 20353; 20354; 20356; 20362; 20371;
576; 20374; 12332; 14721; 14722; 14723; 14724; 20386; 18327; 10375;
10389; 10390; 10391; 10414; 10415; 10416; 14762; 20392; 12363;
20394; 16582; 16583; 20395; 20396; 13747; 20402; 20404; 20405;
6644; 20410; 20416.
[0199] The following SEQ ID NOs correspond to the amino acid
sequences of placenta-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 20843; 375; 20844; 1318; 20849; 20850; 20851; 5925;
20852; 2112; 1330; 20858; 20859; 20860; 20861; 4219; 20865; 20866;
20867; 20868; 20869; 20871; 20872; 20873; 20874; 4239; 4240; 20876;
9680; 9699; 20885; 20886; 20887; 15613; 15614; 15616; 9754; 20890;
20892; 20893; 20894; 20895; 20905; 20906; 488; 20909; 20910; 20911;
20912; 20913; 20914; 20920; 7669; 19509; 20922; 20934; 20938;
18151; 20940; 20945; 20946; 14622; 20947; 9999; 20949; 20951;
10003; 20952; 20953; 20955; 20956; 20958; 20959; 20960; 20962;
20963; 20964; 4409; 20969; 20970; 20971; 20988; 10186; 20991;
20999; 21000; 2283; 21008; 13692; 18284; 4475; 21012; 21014; 21015;
4487; 21019; 21022; 21023; 21024; 3220; 3221; 18319; 21032; 3229;
3230; 3231; 625; 21041; 21042; 21043; 21045; 21046; 21049; 6548;
6549; 6556; 7798; 21053; 19673; 19674; 4577; 14782; 21057; 6599;
21058; 21059; 21060; 21061; 21068; 21069; 21073; 21074; 21078;
21080; 21082; 21083; 21084; 18394; 13776; 16626; 21086; 21087;
21088; 21090; 21091; 19730; 6672; 10706; 21101; 6732; 21107;
21109.
[0200] The following SEQ ID NOs correspond to the amino acid
sequences of prostate-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 16304; 22026; 22027; 13537; 9459; 2100; 9463; 22031;
22032; 22033; 22034; 9472; 9473; 22037; 22038; 22039; 22040; 2989;
22047; 1323; 384; 5935; 22050; 22051; 22052; 22053; 22054; 22055;
2113; 1324; 1325; 1326; 22056; 1327; 22058; 9515; 22064; 5944;
4171; 4172; 3005; 3006; 9526; 15579; 2119; 9528; 22066; 1330; 7568;
397; 22071; 22074; 22085; 22086; 22087; 22088; 19380; 19381; 22089;
22094; 22101; 22102; 22104; 22106; 4219; 1357; 19419; 424; 22113;
22114; 22115; 22116; 22117; 22119; 22120; 20872; 22123; 12223;
22124; 22125; 22126; 22127; 22128; 22132; 15604; 22135; 18058;
22136; 7613; 22137; 22138; 7615; 22140; 9694; 9695; 22144; 22146;
22147; 20885; 20886; 22151; 22152; 22153; 22154; 9736; 6054; 3055;
22159; 1384; 22162; 22163; 1386; 469; 9750; 9751; 22164; 22167;
2184; 22168; 15617; 9786; 14555; 14556; 22171; 22172; 22173; 20890;
22174; 22175; 22176; 6076; 9798; 9804; 22177; 22179; 22180; 22191;
22193; 6088; 6090; 6091; 6092; 6093; 6094; 9875; 22209; 18105;
18106; 1415; 22212; 499; 500; 13631; 22218; 22221; 22222; 7669;
9924; 9925; 1425; 22228; 6135; 7676; 524; 22239; 4345; 4346; 4347;
4348; 4349; 4350; 22240; 15635; 22244; 22247; 22252; 22253; 22254;
22255; 22257; 22259; 22263; 22264; 3148; 6231; 22269; 22287; 22293;
22313; 22318; 22319; 22320; 22324; 22329; 3166; 22342; 3177; 22351;
22357; 22358; 3187; 16505; 22363; 10224; 22365; 22366; 22367;
22368; 22371; 14685; 22373; 22378; 22384; 22387; 22388; 22389;
22390; 6311; 10244; 10245; 22400; 22401; 16530; 14707; 21012;
21014; 17283; 22412; 10287; 22417; 22418; 22419; 22420; 22421;
22422; 22424; 1484; 22430; 22431; 22432; 22433; 22434; 22435;
22436; 3229; 3230; 3231; 22437; 4513; 4521; 22448; 22449; 22450;
22452; 22455; 3237; 3238; 10389; 10390; 10391; 22458; 22459; 22460;
22461; 14757; 14758; 4535; 22464; 22465; 22466; 22467; 22468; 3247;
16565; 7782; 7783; 7784; 7785; 10425; 22474; 22475; 22476; 16567;
16568; 22479; 22480; 10447; 22484; 10467; 22491; 19645; 19647;
19648; 19649; 4565; 10484; 10485; 4566; 4567; 7798; 22494; 4570;
19660; 22496; 14777; 2338; 19665; 10508; 10509; 10511; 16585;
22499; 22500; 22501; 22502; 22503; 10515; 22507; 2353; 22509;
22510; 19684; 3267; 3268; 6611; 22515; 7813; 7814; 10595; 10596;
10598; 22519; 3278; 22526; 22527; 6623; 22530; 22531; 22532; 22533;
22534; 21080; 22547; 16626; 22549; 22551; 22552; 22554; 22559;
22560; 10657; 22562; 10660; 22564; 13065; 22569; 13792; 22571;
22572; 7851; 2380; 22575; 13071; 13794; 13795; 22579; 22581; 22584;
22585; 18424; 18425; 22589; 22590; 22592; 22593; 22594; 22598;
10744; 10745.
[0201] The following SEQ ID NOs correspond to the amino acid
sequences of retina-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 16302; 23606; 23613; 23614; 23615; 23616; 5935; 23617;
4168; 23620; 5961; 12891; 4194; 23630; 23631; 23632; 409; 12914;
12915; 12916; 23637; 23639; 23640; 23641; 23643; 23646; 23647;
23652; 23654; 22125; 2167; 23658; 23663; 9659; 9660; 23664; 22132;
23667; 7614; 23674; 23676; 23677; 23678; 23679; 23680; 23681;
23682; 23683; 23684; 23685; 471; 23688; 23696; 23700; 1417; 22221;
22222; 23712; 23713; 23714; 3095; 23715; 9923; 23721; 14613; 15630;
23724; 18157; 23728; 23729; 23730; 20947; 541; 23738; 23739; 23741;
3148; 23782; 23784; 6296; 23787; 23791; 10199; 1473; 1474; 1475;
1476; 1477; 3198; 3199; 4467; 23799; 23800; 23801; 16522; 6453;
6454; 6455; 6456; 6457; 6458; 6459; 6460; 4478; 23810; 23813;
23814; 23815; 23817; 21022; 23820; 1490; 1491; 23825; 3227; 3228;
23828; 23829; 6515; 15690; 15691; 10383; 10384; 10385; 14756;
12350; 636; 637; 23836; 23837; 23838; 10424; 23839; 23840; 23841;
6546; 23851; 23855; 22484; 23861; 23862; 23863; 23864; 23865;
23866; 23867; 10512; 23878; 23879; 23881; 23884; 14783; 14784;
4587; 23894; 23895; 23899; 19702; 23904; 18405; 23909; 23912;
23913; 23915; 23916; 23919; 2380; 23922; 23924; 19730; 23928;
21109; 2393.
[0202] The following SEQ ID NOs correspond to the amino acid
sequences of salivary gland-specific proteins identified using MPSS
and that have been identified by mass spectrometry as described in
Table 43A: 22031; 22032; 24435; 22056; 24437; 15579; 19371; 24447;
24448; 24450; 16341; 16342; 16343; 16344; 19423; 24456; 24458; 440;
441; 442; 443; 444; 7613; 24463; 470; 24466; 14561; 24499; 24500;
532; 24515; 24516; 4375; 22247; 24519; 24530; 24531; 16507; 6453;
6454; 6455; 6456; 6457; 6458; 6459; 6460; 24533; 24534; 24535;
24536; 24537; 24539; 24540; 24545; 24547; 24548; 10399; 10400;
24550; 24552; 6546; 661; 662; 663; 3257; 13754; 13755; 10595;
10596; 10598; 21069; 21073; 21074; 21078; 18406; 24563; 24564.
[0203] The following SEQ ID NOs correspond to the amino acid
sequences of small intestine-specific proteins identified using
MPSS and that have been identified by mass spectrometry as
described in Table 43A: 5906; 5907; 5908; 5909; 5910; 24919; 24920;
24921; 9485; 2986; 2987; 2988; 16310; 4147; 24928; 2119; 13544;
13545; 24932; 9536; 24933; 24935; 24942; 9574; 24946; 18007; 24963;
24964; 24967; 24968; 9651; 440; 441; 442; 443; 444; 24978; 24979;
24980; 15604; 24985; 22135; 24986; 24987; 9699; 9787; 1394; 24997;
6101; 25001; 9935; 25008; 516; 25009; 14619; 14620; 20946; 14622;
22240; 25011; 19524; 25015; 13653; 25017; 25018; 25024; 25025;
25026; 25027; 25028; 25030; 25031; 25036; 25038; 25043; 25044;
25045; 25046; 25047; 25048; 25049; 25050; 15657; 25055; 6298; 6303;
25064; 13693; 12330; 25068; 25069; 25070; 22437; 6550; 6551; 25076;
25080; 25081; 25086; 25087; 10487; 10488; 19661; 4574; 10502;
25089; 25090; 25092; 25094; 12370; 14791; 16597; 25096; 25104;
25105; 10645; 10646; 25106; 13781; 1568; 1569; 1570; 1571; 1572;
1574; 25112; 25113; 25114; 10699; 10700; 25117; 10735.
[0204] The following SEQ ID NOs correspond to the amino acid
sequences of spinal cord-specific proteins identified using MPSS
and that have been identified by mass spectrometry as described in
Table 43A: 9449; 22031; 22032; 13540; 4139; 4141; 4142; 17978;
2113; 12184; 9550; 9551; 9562; 25488; 25491; 9585; 17209; 17210;
14500; 14501; 14502; 14503; 14504; 25498; 25499; 25500; 424; 3037;
25501; 25502; 25503; 25504; 25505; 22124; 25506; 14523; 4244;
25507; 25508; 1369; 9692; 9746; 9747; 25518; 18072; 18073; 18074;
18075; 18076; 18077; 18078; 18079; 1388; 1389; 25520; 22177; 25521;
18092; 18093; 18094; 25526; 25527; 25528; 19500; 25529; 25531;
25532; 25533; 25534; 18121; 18122; 18123; 18126; 18127; 18129;
18130; 18132; 25535; 25536; 19512; 522; 20354; 25538; 527; 25540;
13653; 4409; 25548; 10161; 25551; 25552; 6297; 25554; 10200; 10201;
10204; 10205; 22365; 22366; 22367; 22368; 25558; 25561; 25562;
25563; 4470; 10248; 13013; 25566; 7742; 14715; 21023; 21024; 25572;
3232; 10375; 10398; 3242; 25577; 25578; 25579; 25581; 25582; 19660;
10512; 25588; 25589; 25593; 25594; 3276; 3277; 25597; 25598; 25602;
25603; 723.
[0205] The following SEQ ID NOs correspond to the amino acid
sequences of spleen-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 26279; 26280; 26281; 26282; 26290; 4135; 26293; 2983;
2984; 2985; 9489; 26296; 9497; 4148; 26299; 26300; 26301; 26304;
5944; 13543; 9522; 9523; 26305; 26306; 26308; 26309; 12890; 26310;
13544; 13545; 26311; 1333; 5961; 26319; 26321; 26322; 26324; 26325;
26329; 26335; 3024; 26339; 26341; 2138; 4219; 9602; 26350; 26351;
9613; 16383; 26355; 26356; 7604; 26357; 26358; 9637; 26362; 16391;
16392; 26363; 26364; 2167; 26365; 19452; 26366; 26370; 26371;
12233; 16401; 26375; 9736; 26380; 22162; 469; 22168; 26381; 9773;
23684; 23685; 3060; 13608; 13609; 1390; 22174; 26383; 26384; 26386;
26390; 26391; 18083; 4280; 4281; 4282; 26400; 26401; 26402; 26409;
14566; 14567; 14568; 14569; 26411; 26420; 13622; 25528; 1417;
26426; 26427; 500; 24499; 7669; 25535; 25536; 26431; 511; 26432;
9939; 26442; 12267; 26444; 26445; 18147; 18148; 18149; 26447;
17246; 26448; 26450; 26451; 26453; 1432; 14620; 532; 26457; 2243;
26463; 14627; 6173; 6174; 6180; 6181; 6182; 6183; 6189; 6190;
10036; 26473; 26474; 26476; 26477; 26478; 20964; 2246; 18175; 2252;
2253; 556; 26503; 2256; 26504; 26505; 20988; 26527; 3167; 10176;
26530; 10186; 26531; 26534; 26538; 26541; 26542; 4455; 26544;
13681; 13682; 26547; 4462; 26561; 3198; 3199; 16522; 4471; 26563;
26564; 26573; 10273; 26574; 26575; 26578; 12332; 4496; 26584;
26585; 26586; 14725; 4498; 1484; 26590; 10327; 3220; 3221; 18318;
14736; 18319; 26598; 26599; 2306; 26603; 6519; 6528; 2312; 26610;
21041; 21042; 21043; 21045; 21046; 26611; 10399; 10400; 26612;
26613; 26614; 26615; 24552; 10410; 19618; 19620; 10413; 26619;
26621; 7782; 7783; 7784; 7785; 12356; 4544; 20392; 4552; 4553;
4554; 23863; 23864; 23865; 23866; 23867; 10489; 7798; 656; 26647;
26649; 23878; 23879; 26650; 10554; 14783; 14784; 1532; 13747;
26657; 26659; 6610; 14793; 26667; 10599; 26673; 26674; 26675; 686;
26680; 18392; 21083; 12397; 3299; 707; 708; 709; 26695; 18404;
26699; 26702; 3308; 3309; 3311; 3312; 26704; 4642; 4643; 10677;
26705; 1583; 16655; 16658; 16659; 16662; 23922; 4655; 26723; 26724;
26725; 26726; 26729; 22589; 26731; 26732; 26739; 6725; 26745;
26746; 26748.
[0206] The following SEQ ID NOs correspond to the amino acid
sequences of stomach-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 13580; 12223; 18058; 27369; 27382; 27383; 27384; 3261;
27385; 1582; 27388; 2386.
[0207] The following SEQ ID NOs correspond to the amino acid
sequences of testis-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 28427; 28433; 5922; 28437; 28438; 28439; 28442; 16310;
22047; 28447; 28452; 28453; 28458; 28459; 28461; 5943; 28465;
13543; 28466; 28467; 28468; 28469; 13544; 13545; 28480; 28483;
28484; 19371; 399; 400; 28495; 28496; 28497; 28501; 28504; 28513;
28520; 12897; 12898; 12902; 12903; 12904; 12908; 28534; 28541;
28542; 28548; 28564; 28566; 28568; 28569; 28570; 28573; 22104;
28576; 28577; 28580; 3033; 28581; 9603; 4226; 28595; 20869; 28602;
1358; 28608; 23646; 23647; 9637; 26362; 28614; 28615; 14523; 28622;
28625; 28626; 28627; 19453; 19454; 28634; 28635; 28636; 28637;
28640; 28642; 28646; 28651; 22144; 22146; 22147; 28656; 28659;
28660; 1383; 20320; 20321; 20322; 28675; 28676; 9754; 2184; 22168;
28681; 28682; 9787; 28699; 28702; 28703; 28707; 28710; 1402; 28729;
28738; 28748; 28751; 28752; 28755; 28756; 28757; 28759; 28771;
28777; 28781; 28784; 28787; 4314; 28794; 1425; 28800; 4323; 6126;
17243; 9941; 9942; 9943; 9944; 28810; 28811; 3099; 28816; 28817;
28818; 14616; 14617; 14619; 28819; 28820; 28821; 20945; 15634;
9981; 9982; 28832; 28833; 10005; 28845; 28846; 14627; 12276; 12277;
28853; 28854; 28856; 3139; 28858; 28859; 6224; 28867; 28870; 28871;
3148; 28876; 28878; 28879; 28894; 28921; 28931; 28934; 22287;
28939; 28940; 28941; 28942; 28943; 28959; 28960; 28961; 28974;
28999; 29002; 29036; 29037; 22313; 22318; 22319; 22320; 22324;
29055; 29057; 29058; 29059; 29060; 29061; 29062; 29063; 29064;
29065; 22329; 1447; 29074; 29075; 25055; 29077; 29078; 29085; 3172;
19565; 19566; 29086; 12306; 29088; 29089; 29092; 13007; 29101;
29109; 14681; 29121; 4467; 16522; 13695; 13696; 3204; 18289; 10273;
10279; 10280; 10281; 29125; 10285; 10286; 29126; 29128; 29129;
29130; 29131; 29132; 29141; 10298; 29159; 10316; 10324; 6493; 6494;
29182; 14735; 29185; 3227; 3228; 10338; 15683; 29195; 29207; 29210;
29217; 3241; 29225; 29226; 29227; 3245; 10426; 29231; 22476; 639;
12356; 4544; 642; 29239; 23861; 23862; 29257; 29258; 29261; 29262;
29263; 10476; 19654; 19655; 19656; 19659; 659; 18366; 3257; 1536;
1537; 29286; 10564; 22509; 22510; 25593; 29290; 26667; 29298;
29299; 16603; 29301; 29302; 673; 674; 675; 2357; 2358; 676; 29314;
29319; 3276; 3277; 29324; 29328; 6624; 29333; 22533; 29344; 12397;
29354; 29355; 29363; 29364; 29365; 13065; 29368; 29376; 10677;
7851; 4649; 1582; 29387; 1583; 29388; 29391; 4655; 29394; 29395;
723; 22589; 12411; 1590; 29413; 29417; 29419; 29423; 29424; 29425;
7871.
[0208] The following SEQ ID NOs correspond to the amino acid
sequences of thymus-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 19351; 14455; 22031; 22032; 30730; 1324; 1325; 1326;
22056; 30734; 13543; 9528; 30736; 30737; 4177; 1333; 30738; 12895;
30742; 28580; 30746; 30747; 30748; 30749; 30753; 4226; 28602;
23643; 30757; 23658; 6019; 30759; 30760; 30761; 30762; 6026; 16401;
470; 23684; 23685; 9786; 9787; 30770; 30771; 20905; 30779; 30783;
30784; 488; 6088; 6090; 6091; 6092; 6093; 6094; 9883; 26426; 26427;
30790; 500; 30793; 30795; 4323; 30796; 30797; 30798; 9952; 522;
26444; 26445; 9981; 9982; 30803; 30809; 28845; 28846; 14627; 6191;
30813; 30816; 6225; 6226; 30817; 30818; 30822; 30823; 30824; 30825;
30826; 30887; 30888; 30889; 30890; 26530; 30892; 30893; 3187;
20999; 7729; 4455; 26544; 30898; 30899; 25561; 19572; 30902; 26561;
12315; 12316; 3198; 3199; 4471; 26564; 30903; 16535; 30904; 7742;
30905; 30906; 30907; 30908; 30911; 30914; 30915; 30916; 7767;
29210; 30920; 30921; 30922; 19618; 19620; 23841; 30928; 30929;
6548; 6549; 22479; 18342; 23863; 23864; 23865; 23866; 23867; 29257;
3255; 30935; 30936; 30937; 19663; 19664; 30938; 6585; 30939; 30940;
30943; 30944; 30946; 4588; 4590; 14793; 30954; 30955; 30956; 27385;
30959; 30960; 3276; 3277; 30961; 10639; 30962; 16627; 30968; 30969;
10660; 18404; 13065; 3308; 3309; 3311; 3312; 30974; 30975; 30976;
30977; 30978; 13794; 13795; 30981; 10701; 30982; 30983; 30984;
30985; 30992.
[0209] The following SEQ ID NOs correspond to the amino acid
sequences of thyroid-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 31511; 31515; 31516; 31517; 31518; 3001; 31520; 31529;
31530; 31531; 16341; 16342; 16343; 16344; 24963; 9611; 14517;
31537; 23679; 9744; 31544; 9786; 7627; 7628; 9797; 31546; 1400;
1401; 31547; 18111; 31564; 31565; 31566; 2242; 31572; 19523; 31576;
16458; 31582; 31583; 20969; 31584; 22269; 1453; 1454; 31589; 31590;
31593; 31596; 31597; 31598; 31599; 31601; 31602; 14699; 14715;
31612; 31613; 31614; 31615; 31616; 10327; 18314; 23825; 1495;
18319; 6506; 4510; 10347; 31620; 31632; 2342; 2343; 2344; 2345;
10560; 31638; 31642; 6626; 6628; 31646; 14835; 31652; 31654; 717;
31658; 31670; 31671; 31672.
[0210] The following SEQ ID NOs correspond to the amino acid
sequences of trachea-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 31889; 17979; 17980; 17981; 31891; 3025; 9602; 7613;
31897; 31898; 9795; 3071; 31902; 16458; 2269; 2271; 31920; 4475;
4487; 22418; 19618; 19620; 31925; 31926; 31927; 31928; 25096; 6619;
6620; 4605; 12395.
[0211] The following SEQ ID NOs correspond to the amino acid
sequences of uterus-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 43A: 32066; 32067; 32068; 32069; 32075; 32079; 9608; 32081;
32082; 32083; 32084; 22218; 9931; 32090; 32092; 32093; 9999; 20949;
10044; 32096; 16495; 22342; 32107; 32108; 32111; 32112; 32114;
19606; 10347; 32115; 32116; 32121; 32122; 23863; 23864; 23865;
23866; 23867; 13740; 19673; 19674; 26667; 3273; 31646; 1568; 1569;
1570; 1571; 1572; 1574; 6732.
[0212] The following SEQ ID NOs correspond to the amino acid
sequences of prostate-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 44A: 16304; 22026; 22027; 13537; 9459; 2100; 9463; 22031;
22032; 22033; 22034; 9472; 9473; 22037; 22038; 22039; 22040; 32221;
2989; 22047; 383; 1323; 384; 5935; 22050; 22051; 22052; 22053;
22054; 22055; 2113; 1324; 1325; 1326; 22056; 1327; 22058; 9515;
22064; 5944; 4171; 4172; 3005; 3006; 9526; 15579; 2119; 9528;
22066; 1330; 7568; 397; 22071; 22074; 22085; 22086; 22087; 22088;
19380; 19381; 22089; 22094; 22101; 22102; 22104; 22106; 4219; 1357;
19419; 424; 22113; 22114; 22115; 22116; 22117; 22119; 22120; 20872;
22123; 12223; 22124; 22125; 22126; 22127; 22128; 22132; 15604;
22135; 18058; 22136; 7613; 22137; 22138; 7615; 22140; 9694; 9695;
22144; 22146; 22147; 20885; 20886; 22151; 22152; 22153; 22154;
9736; 6054; 3055; 22159; 1384; 22162; 22163; 1386; 469; 9750; 9751;
22164; 22167; 2184; 22168; 15617; 9786; 14555; 14556; 22171; 22172;
22173; 20890; 22174; 22175; 22176; 6076; 9798; 9804; 22177; 22179;
14561; 22180; 22191; 22193; 1404; 6088; 6090; 6091; 6092; 6093;
6094; 9875; 22209; 18105; 18106; 1415; 22212; 499; 500; 13631;
22218; 22221; 22222; 7669; 9924; 9925; 1425; 22228; 6135; 7676;
524; 22239; 4345; 4346; 4347; 4348; 4349; 4350; 22240; 15635;
22244; 22247; 22252; 22253; 22254; 22255; 22257; 22259; 22263;
22264; 3148; 6231; 22269; 22287; 22293; 22313; 22318; 22319; 22320;
22324; 22329; 3166; 22342; 3177; 22351; 22357; 22358; 3187; 16505;
22363; 10224; 22365; 22366; 22367; 22368; 22371; 14685; 22373;
32227; 22378; 22384; 22387; 22388; 22389; 22390; 6311; 10244;
10245; 22400; 22401; 16530; 18289; 14707; 21012; 21014; 17283;
22412; 10287; 22417; 22418; 22419; 22420; 22421; 22422; 22424;
1484; 22430; 22431; 32228; 22432; 22433; 32229; 22434; 22435;
22436; 3229; 3230; 3231; 22437; 4513; 4521; 22448; 22449; 22450;
22452; 22455; 3237; 3238; 10389; 10390; 10391; 22458; 22459; 22460;
22461; 14757; 14758; 4535; 22464; 22465; 22466; 22467; 22468; 3247;
16565; 7782; 7783; 7784; 7785; 10425; 22474; 22475; 22476; 16567;
16568; 22479; 22480; 10447; 22484; 10467; 22491; 19645; 19647;
19648; 19649; 4565; 10484; 10485; 4566; 4567; 7798; 22494; 4570;
19660; 22496; 14777; 2338; 19665; 10508; 10509; 10511; 16585;
22499; 22500; 22501; 22502; 22503; 10515; 22507; 2353; 22509;
22510; 19684; 3267; 3268; 6611; 22515; 7813; 7814; 10595; 10596;
10598; 22519; 25594; 3278; 22526; 22527; 6623; 22530; 22531; 22532;
22533; 22534; 10636; 21080; 22547; 16626; 22549; 22551; 22552;
22554; 22559; 22560; 10657; 22562; 10660; 22564; 13065; 22569;
13792; 22571; 22572; 7851; 2380; 22575; 13071; 13794; 13795; 22579;
22581; 22584; 22585; 18424; 18425; 22589; 22590; 22592; 22593;
22594; 22598; 10744; 10745.
[0213] The following SEQ ID NOs correspond to the amino acid
sequences of testis-specific proteins identified using MPSS and
that have also been identified by mass spectrometry as described in
Table 44A: 28427; 28433; 32274; 5922; 28437; 28438; 28439; 28442;
16310; 22047; 28447; 28452; 28453; 28458; 28459; 28461; 5943;
28465; 13543; 28466; 28467; 28468; 28469; 13544; 13545; 28480;
28483; 28484; 19371; 399; 400; 28495; 28496; 28497; 28501; 28504;
28513; 28520; 12897; 12898; 12902; 12903; 12904; 12908; 28534;
28541; 28542; 28548; 28564; 28566; 28568; 28569; 28570; 28573;
22104; 28576; 28577; 28580; 3033; 28581; 7601; 9603; 4226; 28595;
20869; 28602; 1358; 28608; 23646; 23647; 9637; 26362; 28614; 28615;
1364; 14523; 28622; 32277; 28625; 28626; 28627; 19453; 19454;
28634; 28635; 28636; 28637; 28640; 28642; 28646; 28651; 22144;
22146; 22147; 28656; 28659; 28660; 1383; 20320; 20321; 20322;
28675; 28676; 9754; 2184; 22168; 28681; 28682; 9787; 28699; 28702;
28703; 28707; 28710; 1402; 28729; 28738; 28748; 28751; 28752;
28755; 28756; 28757; 28759; 28771; 13622; 28777; 28781; 28784;
28787; 4314; 28794; 1425; 28800; 4323; 6126; 17243; 9941; 9942;
9943; 9944; 28810; 28811; 3099; 28816; 28817; 28818; 14616; 14617;
14619; 28819; 28820; 28821; 20945; 15634; 9981; 9982; 28832; 28833;
10005; 28845; 28846; 14627; 32278; 12276; 12277; 28853; 28854;
28856; 3139; 28858; 28859; 6224; 28867; 28870; 28871; 3148; 28876;
28878; 28879; 28894; 28921; 28931; 28934; 22287; 28939; 28940;
28941; 28942; 28943; 28959; 28960; 28961; 28974; 28999; 29002;
29036; 29037; 22313; 22318; 22319; 22320; 22324; 29055; 29057;
29058; 29059; 29060; 29061; 29062; 29063; 29064; 29065; 22329;
32283; 1447; 29074; 29075; 25055; 29077; 29078; 29085; 3172; 19565;
19566; 29086; 12306; 29088; 29089; 29092; 13007; 29101; 29109;
14681; 32284; 29121; 4467; 16522; 13695; 13696; 3204; 18289; 10273;
10279; 10280; 10281; 29125; 10285; 10286; 29126; 29128; 29129;
29130; 29131; 29132; 29141; 10298; 29159; 10316; 10324; 6493; 6494;
29182; 14735; 29185; 3227; 3228; 10338; 15683; 29195; 29207; 29210;
29217; 3241; 29225; 29226; 29227; 3245; 10426; 29231; 22476; 639;
12356; 4544; 642; 29239; 23861; 23862; 29257; 29258; 29261; 29262;
29263; 10476; 19654; 19655; 19656; 19659; 659; 18366; 3257; 1536;
1537; 29286; 10564; 22509; 22510; 25593; 29290; 26667; 29298;
29299; 16603; 29301; 29302; 673; 674; 675; 2357; 2358; 676; 29314;
29319; 3276; 3277; 29324; 29328; 6624; 29333; 32286; 32287; 22533;
29344; 12397; 29354; 29355; 29363; 29364; 29365; 13065; 29368;
29376; 10677; 7851; 4649; 1582; 29387; 1583; 29388; 29391; 4655;
29394; 29395; 723; 22589; 10724; 10725; 12411; 1590; 29413; 29417;
29419; 29423; 29424; 29425; 7871.
[0214] The following SEQ ID NOs correspond to the amino acid
sequences of mammary gland-specific proteins identified using MPSS
and that have been identified by mass spectrometry as described in
Table 44A: 17184; 17185; 17186; 17187; 17188; 7558; 17189; 17190;
17191; 5935; 5941; 5942; 17196; 17197; 17198; 32330; 32332; 32333;
32334; 32335; 17200; 17201; 408; 3025; 17209; 17210; 17211; 17212;
17213; 17214; 17218; 17219; 17220; 3055; 3056; 3057; 3058; 9786;
17228; 6088; 6090; 6091; 6092; 6093; 6094; 32339; 499; 12250;
12251; 17243; 518; 519; 17246; 532; 17258; 17259; 17261; 17262;
17263; 3171; 17275; 3191; 17281; 17283; 17284; 29125; 10298; 17288;
32229; 17292; 17293; 17298; 17301; 17302; 17303; 17305; 17306;
17307; 1519; 13740; 10486; 16574; 10491; 17312; 17313; 17314;
17315; 2342; 2343; 2344; 2345; 16587; 6610; 13051; 16622; 14821;
17326; 1564; 17329; 17332; 14835; 17333; 7846; 13065; 17335; 1582;
6672; 17336; 723.
[0215] The following SEQ ID NOs correspond to the amino acid
sequences of uterus-specific proteins identified using MPSS and
that have been identified by mass spectrometry as described in
Table 44A: 32066; 32067; 5925; 32068; 32069; 32075; 32079; 9608;
32081; 32082; 32083; 32084; 22218; 9931; 32090; 32092; 32093; 9999;
20949; 10044; 32096; 16495; 22342; 32107; 32108; 32111; 32112;
32114; 19606; 10347; 32115; 32116; 32121; 32122; 23863; 23864;
23865; 23866; 23867; 13740; 19673; 19674; 26667; 3273; 21080;
31646; 1568; 1569; 1570; 1571; 1572; 1574; 6732.
[0216] The following SEQ ID NOs correspond to the amino acid
sequences of CL1 (late-stage prostate cancer cell line)-specific
proteins identified by MPSS that have also been identified by mass
spectrometry as described in Table 45A: 32481; 32482; 32483; 380;
381; 5944; 32486; 9527; 5961; 4178; 32490; 32491; 2125; 3019; 3020;
32495; 32496; 26339; 32503; 32506; 12221; 12222; 14523; 32511;
32512; 469; 32513; 1388; 1389; 32514; 22179; 32517; 13630; 32523;
32524; 6135; 7676; 32529; 32530; 28833; 19524; 7690; 6284; 22342;
10186; 22351; 32539; 32540; 32541; 32543; 13685; 13686; 32547;
26574; 26575; 13019; 13020; 22419; 22420; 22421; 32550; 14735;
32228; 6501; 32554; 32555; 32556; 32557; 32560; 32561; 2337; 32563;
19668; 7804; 32565; 14790; 16597; 678; 21068; 32568; 2372; 699;
4638; 32574; 32575; 32576; 32577; 22589; 32580; 32581.
[0217] The following SEQ ID NOs correspond to the amino acid
sequences of LNCaP (early-stage prostate cancer cell line)-specific
proteins identified by MPSS that have also been identified by mass
spectrometry as described in Table 45A: 22054; 32817; 32818; 32823;
426; 427; 428; 6030; 1390; 25527; 19500; 499; 32828; 32829; 32830;
32831; 32832; 19512; 1432; 32834; 32835; 32836; 32841; 10200;
10201; 10204; 10205; 32848; 2296; 2297; 32852; 30911; 6496; 3229;
3230; 3231; 32859; 14764; 32866.
[0218] The following SEQ ID NOs correspond to the amino acid
sequences of normal prostate-specific proteins identified by MPSS
that have also been identified by mass spectrometry as described in
Table 45A: 16304; 22026; 22027; 13537; 9459; 2100; 9463; 22031;
22032; 22033; 22034; 9472; 9473; 22037; 22038; 22039; 22040; 2989;
22047; 383; 1323; 384; 5935; 22050; 22051; 22052; 22053; 22054;
22055; 2113; 1324; 1325; 1326; 22056; 1327; 22058; 9515; 22064;
5944; 4171; 4172; 3006; 9526; 15579; 2119; 9528; 22066; 1330; 7568;
397; 22071; 22074; 22085; 22086; 22087; 22088; 19380; 19381; 22089;
22094; 22101; 22102; 22104; 4219; 1357; 19419; 424; 22113; 22114;
22115; 22116; 22117; 22119; 22120; 20872; 22123; 12223; 22124;
22125; 22126; 22127; 22128; 15604; 22135; 18058; 22136; 7613;
22137; 22138; 7615; 22140; 9694; 9695; 22144; 22146; 22147; 20885;
20886; 22151; 22152; 22153; 22154; 9736; 6054; 3055; 22159; 1384;
22162; 22163; 1386; 469; 9750; 9751; 22164; 22167; 2184; 22168;
15617; 9786; 14555; 14556; 22171; 22172; 22173; 20890; 22174;
22175; 22176; 6076; 9798; 9804; 22177; 22179; 14561; 22180; 22193;
6088; 6090; 6091; 6092; 6093; 6094; 9875; 22209; 18105; 18106;
1415; 22212; 499; 500; 13631; 22218; 22221; 22222; 7669; 9924;
9925; 1425; 22228; 6135; 7676; 524; 22239; 4345; 4346; 4347; 4348;
4349; 4350; 22240; 15635; 22244; 22247; 22252; 22253; 22254; 22255;
22257; 22259; 22263; 22264; 3148; 6231; 22269; 22287; 22293; 22313;
22318; 22319; 22320; 22324; 22329; 3166; 22342; 3177; 22351; 22357;
22358; 3187; 16505; 22363; 10224; 22365; 22366; 22367; 22368;
22371; 14685; 22373; 22378; 22384; 22387; 22388; 22389; 22390;
6311; 10244; 10245; 22400; 22401; 16530; 18289; 14707; 21012;
21014; 17283; 22412; 10287; 22417; 22418; 22419; 22420; 22421;
22422; 1484; 22430; 22431; 32228; 22432; 22433; 32229; 22434;
22435; 22436; 22437; 4513; 4521; 22448; 22449; 22450; 22452; 22455;
3237; 3238; 10389; 10390; 10391; 22458; 22459; 22460; 22461; 14757;
14758; 4535; 22464; 22465; 22466; 22467; 22468; 3247; 16565; 7782;
7783; 7784; 7785; 10425; 22474; 22476; 16567; 16568; 22479; 22480;
10447; 22484; 10467; 22491; 19645; 19647; 19648; 19649; 4565; 4566;
4567; 7798; 22494; 4570; 19660; 22496; 14777; 2338; 19665; 10508;
10509; 10511; 16585; 22499; 22500; 22501; 22502; 22503; 10515;
22507; 22509; 22510; 19684; 3267; 3268; 6611; 22515; 7813; 7814;
10595; 10596; 10598; 22519; 25594; 3278; 22526; 22527; 6623; 22530;
22531; 22532; 10636; 21080; 22547; 16626; 22549; 22551; 22552;
22554; 22559; 22560; 10657; 22562; 10660; 22564; 13065; 22569;
13792; 22571; 22572; 7851; 22575; 13071; 13794; 13795; 22579;
22581; 22584; 22585; 18424; 18425; 22589; 22590; 22592; 22593;
22594; 22598; 10744; 10745.
[0219] The following SEQ ID NOs correspond to the polynucleotides
encoding adrenal gland-specific proteins as described in Table 47A
identified using SBS: 52865; 20; 52866; 27630; 77; 78; 79; 80; 81;
52867; 91; 20175; 20176; 52868; 111; 112; 152; 153; 30543; 30544;
30545; 173; 52869; 52870; 52871; 52872; 52873; 206; 207; 52874;
235; 280; 281; 52875; 52876; 312; 313; 52877; 52878; 52879.
[0220] The following SEQ ID NOs correspond to the amino acid
sequences of adrenal gland-specific proteins as described in Table
47A identified using SBS: 52880; 388; 52881; 28632; 445; 446; 447;
448; 449; 52882; 459; 20316; 20317; 52883; 479; 480; 520; 521;
30806; 30807; 30808; 541; 52884; 52885; 52886; 52887; 52888; 574;
575; 52889; 603; 649; 648; 52890; 52891; 680; 681; 52892; 52893;
52894.
[0221] The following SEQ ID NO corresponds to the polynucleotide
encoding an artery-specific protein as described in Table 48A
identified using SBS: 24329.
[0222] The following SEQ ID NO correspond to the amino acid
sequence of an artery-specific protein as described in Table 48A
identified using SBS: 24459.
[0223] The following SEQ ID NOs correspond to the polynucleotides
encoding bladder-specific proteins as described in Table 49A
identified using SBS: 1032; 52986; 52987; 52988; 52989; 52990;
21541; 52991; 52992; 52993; 52994; 1187; 1188; 52995; 52996; 52997;
52998; 1259; 1260; 1261; 52999; 53000; 1301; 4105.
[0224] The following SEQ ID NOs correspond to the amino acid
sequences of bladder-specific proteins as described in Table 49A
identified using SBS: 1315; 53001; 53002; 53003; 53004; 53005;
22128; 53006; 53007; 53008; 53009; 1470; 1471; 53010; 53011; 53012;
53013; 1542; 1543; 1544; 53014; 53015; 1584; 4654.
[0225] The following SEQ ID NOs correspond to the polynucleotides
encoding brain-specific proteins as described in Table 50A
identified using SBS: 1796; 1797; 1798; 53036; 53037; 53038; 53039;
53040; 53041; 53042; 53043; 8142; 53044; 53045; 53046; 53047;
11914; 11915; 53048; 53049; 53050; 5100; 12673; 12674; 12675;
12676; 12677; 53051; 53052; 53053; 53054; 11919; 11920; 25344;
25345; 53055; 53056; 23286; 53057; 3605; 3606; 3607; 15916; 3608;
53058; 2643; 53059; 8191; 3620; 3621; 5112; 5113; 2646; 2647; 5115;
5116; 53060; 53061; 7236; 7237; 53062; 53063; 8201; 53064; 2651;
53065; 17519; 53066; 2652; 53067; 5128; 1051; 53068; 53069; 53070;
53071; 53072; 53073; 53074; 53075; 25349; 53076; 53077; 53078;
53079; 2667; 53080; 53081; 53082; 53083; 8262; 3659; 53084; 5153;
5154; 5155; 5158; 53085; 53086; 53087; 3660; 3661; 3662; 3663;
3664; 3665; 3666; 3667; 53088; 21515; 53089; 53090; 53091; 53092;
19012; 53093; 53094; 53095; 53096; 53097; 3681; 2682; 2683; 53098;
53099; 53100; 53101; 7275; 53102; 32018; 53103; 8312; 25364; 53104;
8318; 19032; 19033; 53105; 2691; 53106; 53107; 53108; 8334; 53109;
8337; 7282; 53110; 53111; 53112; 53113; 8354; 53114; 53115; 53116;
8378; 8381; 8382; 53117; 20178; 53118; 53119; 53120; 53121; 3711;
53122; 53123; 53124; 32769; 32770; 32771; 3712; 53125; 53126;
53127; 5223; 5226; 5227; 5229; 5230; 53128; 53129; 53130; 53131;
8455; 53132; 1107; 1108; 2714; 53133; 53134; 53135; 53136; 25926;
14146; 8481; 8482; 11985; 31386; 31387; 31388; 31389; 31390; 31391;
31392; 31394; 31395; 53137; 25382; 53138; 53139; 53140; 7316; 7317;
53141; 53142; 53143; 8524; 53144; 53145; 53146; 8536; 8540; 53147;
53148; 53149; 53150; 53151; 53152; 53153; 8549; 8550; 53154; 53155;
3752; 3753; 3754; 3755; 2725; 53156; 53157; 8556; 7332; 24800;
2730; 2731; 53158; 53159; 53160; 53161; 53162; 53163; 21627; 21628;
21629; 21630; 53164; 7338; 2737; 3768; 5272; 53165; 8579; 8580;
8581; 8582; 8583; 53166; 53167; 53168; 8586; 53169; 53170; 8587;
8588; 53171; 53172; 53173; 53174; 53175; 53176; 53177; 3787; 53178;
53179; 27818; 27819; 53180; 53181; 53182; 53183; 12014; 2749; 3804;
3805; 3806; 8668; 8669; 53184; 53185; 5311; 5312; 5313; 5314; 5315;
5316; 53186; 53187; 53188; 53189; 8670; 8671; 53190; 53191; 53192;
53193; 53194; 2752; 53195; 53196; 19119; 27845; 27846; 53197;
53198; 8691; 8692; 53199; 53200; 53201; 53202; 53203; 53204; 2784;
53205; 53206; 5379; 5380; 5381; 5382; 5383; 5384; 5385; 5386; 5387;
5388; 5389; 2785; 2789; 53207; 53208; 21681; 53209; 53210; 53211;
53212; 53213; 53214; 53215; 53216; 53217; 53218; 53219; 53220;
53221; 53222; 53223; 53224; 30591; 53225; 53226; 5434; 53227;
53228; 53229; 53230; 53231; 53232; 53233; 53234; 53235; 53236;
53237; 5435; 53238; 53239; 53240; 53241; 53242; 53243; 53244;
53245; 53246; 53247; 28013; 53248; 53249; 53250; 53251; 53252;
53253; 53254; 30618; 53255; 53256; 53257; 53258; 53259; 3892;
53260; 23449; 53261; 53262; 28072; 53263; 53264; 53265; 53266;
53267; 53268; 53269; 53270; 3899; 12796; 12797; 53271; 53272;
53273; 53274; 53275; 14253; 53276; 3902; 53277; 53278; 53279; 7391;
7392; 7393; 7394; 53280; 53281; 53282; 53283; 53284; 53285; 53286;
53287; 53288; 53289; 53290; 53291; 53292; 12053; 8884; 8885; 53293;
53294; 53295; 53296; 53297; 3907; 3908; 3909; 53298; 53299; 53300;
53301; 2836; 8912; 53302; 8914; 3917; 12061; 53303; 53304; 53305;
53306; 53307; 32795; 53308; 53309; 53310; 53311; 8934; 8935; 20742;
20743; 53312; 7408; 1199; 53313; 53314; 53315; 2849; 12806; 53316;
12807; 3933; 5636; 5637; 25426; 12068; 8949; 53317; 53318; 8950;
53319; 3936; 2860; 5640; 53320; 53321; 3938; 23483; 23484; 3941;
3942; 53322; 53323; 53324; 53325; 53326; 53327; 53328; 53329;
53330; 53331; 53332; 53333; 53334; 17128; 53335; 53336; 244; 245;
20245; 53337; 53338; 3954; 28182; 53339; 14324; 12820; 7428; 7429;
12088; 53340; 53341; 53342; 53343; 3974; 3975; 16154; 16155; 16156;
16157; 16158; 16159; 5691; 5692; 2878; 53344; 53345; 53346; 53347;
53348; 2887; 2888; 3987; 53349; 53350; 23506; 53351; 53352; 12099;
53353; 53354; 53355; 53356; 53357; 53358; 3994; 53359; 23525;
23526; 23527; 16175; 53360; 5739; 53361; 53362; 53363; 53364;
53365; 20252; 28267; 53366; 53367; 23542; 53368; 53369; 53370;
9166; 9167; 53371; 12107; 5752; 53372; 53373; 53374; 53375; 53376;
53377; 53378; 53379; 53380; 53381; 53382; 53383; 53384; 53385;
53386; 53387; 53388; 53389; 28284; 12836; 53390; 300; 53391; 53392;
53393; 23559; 4035; 12839; 5774; 53394; 53395; 53396; 53397; 53398;
20258; 20259; 2914; 53399; 53400; 53401; 53402; 28300; 53403; 5781;
25454; 53404; 9291; 14396; 53405; 53406; 4059; 4060; 4061; 53407;
53408; 53409; 53410; 53411; 53412; 53413; 53414; 53415; 53416;
53417; 5795; 53418; 2927; 4066; 5796; 9301; 53419; 53420; 53421;
53422; 4069; 4070; 4071; 53423; 7509; 53424; 53425; 53426; 19306;
7515; 2941; 12142; 53427; 4081; 53428; 53429; 2951; 53430; 5812;
5813; 5814; 5815; 5816; 5817; 5818; 5819; 53431; 53432; 1294; 4092;
53433; 21988; 53434; 5825; 2958; 5826; 2959; 5827; 53435; 53436;
53437; 53438; 53439; 5884; 53440; 2973.
[0226] The following SEQ ID NOs correspond to the amino acid
sequences of brain-specific proteins as described in Table 50A
identified using SBS: 2095; 2096; 2097; 53441; 53442; 53443; 53444;
53445; 53446; 53447; 53448; 9470; 53449; 53450; 53451; 53452;
12170; 12171; 53453; 53454; 53455; 5934; 12881; 12882; 12883;
12884; 12885; 53456; 53457; 53458; 53459; 12175; 12176; 25484;
25485; 53460; 53461; 23617; 53462; 4154; 4157; 4156; 16313; 4155;
53463; 2999; 53464; 9519; 4169; 4170; 5946; 5947; 3002; 3003; 5949;
5950; 53465; 53466; 7566; 7567; 53467; 53468; 9529; 53469; 3007;
53470; 17988; 53471; 3008; 53472; 5962; 1334; 53473; 53474; 53475;
53476; 53477; 53478; 53479; 53480; 25489; 53481; 53482; 53483;
53484; 3023; 53485; 53486; 53487; 53488; 9590; 4208; 53489; 5987;
5988; 5989; 5992; 53490; 53491; 53492; 4209; 4210; 4211; 4212;
4213; 4214; 4215; 4216; 53493; 22102; 53494; 53495; 53496; 53497;
19416; 53498; 53499; 53500; 53501; 53502; 4230; 3039; 3038; 53503;
53504; 53505; 53506; 7605; 53507; 32081; 53508; 9640; 25504; 53509;
9646; 19437; 19436; 53510; 3047; 53511; 53512; 53513; 9662; 53514;
9665; 7612; 53515; 53516; 53517; 53518; 9682; 53519; 53520; 53521;
9706; 9709; 9710; 53522; 20319; 53523; 53524; 53525; 53526; 4260;
53527; 53528; 53529; 32825; 32826; 32827; 4261; 53530; 53531;
53532; 6057; 6060; 6061; 6063; 6064; 53533; 53534; 53535; 53536;
9783; 53537; 1390; 1391; 3070; 53538; 53539; 53540; 53541; 26396;
14560; 9809; 9810; 12241; 31548; 31549; 31550; 31551; 31552; 31553;
31554; 31556; 31557; 53542; 25522; 53543; 53544; 53545; 7646; 7647;
53546; 53547; 53548; 9852; 53549; 53550; 53551; 9864; 9868; 53552;
53553; 53554; 53555; 53556; 53557; 53558; 9877; 9878; 53559; 53560;
4301; 4302; 4303; 4304; 3081; 53561; 53562; 9884; 7662; 25003;
3086; 3087; 53563; 53564; 53565; 53566; 53567; 53568; 22214; 22215;
22216; 22217; 53569; 7668; 3093; 4317; 6106; 53570; 9907; 9908;
9909; 9910; 9911; 53571; 53572; 53573; 9914; 53574; 53575; 9915;
9916; 53576; 53577; 53578; 53579; 53580; 53581; 53582; 4336; 53583;
53584; 28820; 28821; 53585; 53586; 53587; 53588; 12270; 3105; 4353;
4354; 4355; 9996; 9997; 53589; 53590; 6145; 6149; 6147; 6148; 6146;
6150; 53591; 53592; 53593; 53594; 9998; 9999; 53595; 53596; 53597;
53598; 53599; 3108; 53600; 53601; 19523; 28848; 28847; 53602;
53603; 10019; 10020; 53604; 53605; 53606; 53607; 53608; 53609;
3140; 53610; 53611; 6213; 6214; 6215; 6216; 6221; 6218; 6219; 6220;
6217; 6222; 6223; 3141; 3145; 53612; 53613; 22268; 53614; 53615;
53616; 53617; 53618; 53619; 53620; 53621; 53622; 53623; 53624;
53625; 53626; 53627; 53628; 53629; 30854; 53630; 53631; 6268; 6269;
53632; 53633; 53634; 53635; 53636; 53637; 53638; 53639; 53640;
53641; 53642; 53643; 53644; 53645; 53646; 53647; 53648; 53649;
53650; 53651; 53652; 29015; 53653; 53654; 53655; 53656; 53657;
53658; 53659; 30881; 53660; 53661; 53662; 53663; 53664; 4441;
53665; 23780; 53666; 53667; 29074; 53668; 53669; 53670; 53671;
53672; 53673; 53674; 53675; 4448; 13004; 13005; 53676; 53677;
53678; 53679; 53680; 14667; 53681; 4451; 53682; 53683; 53684; 7721;
7722; 7723; 7724; 53685; 53686; 53687; 53688; 53689; 53690; 53691;
53692; 53693; 53694; 53695; 53696; 53697; 12309; 10212; 10213;
53698; 53699; 53700; 53701; 53702; 4456; 4457; 4458; 53703; 53704;
53705; 53706; 3192; 10240; 53707; 10242; 4466; 12317; 53708; 53709;
53710; 53711; 53712; 32851; 53713; 53714; 53715; 53716; 10262;
10263; 21009; 21010; 53717; 7738; 1482; 53718; 53719; 53720; 3205;
13014; 53721; 13015; 4482; 6470; 6471; 25566; 12324; 10277; 53722;
53723; 10278; 53724; 4485; 3216; 6474; 53725; 53726; 4487; 23814;
23815; 4490; 4491; 53727; 53728; 53729; 53730; 53731; 53732; 53733;
53734; 53735; 53736; 53737; 53738; 53739; 17288; 53740; 53741; 612;
613; 20386; 53742; 53743; 4503; 29184; 53744; 14738; 13028; 7758;
7759; 12344; 53745; 53746; 53747; 53748; 4523; 4524; 16552; 16553;
16551; 16554; 16555; 16556; 6525; 6526; 3234; 53749; 53750; 53751;
53752; 53753; 3244; 3243; 4536; 53754; 53755; 23837; 53756; 53757;
12355; 53758; 53759; 53760; 53761; 53762; 53763; 4543; 53764;
23856; 23857; 23858; 16572; 53765; 6573; 53766; 53767; 53768;
53769; 53770; 20393; 29269; 53771; 53772; 23873; 53773; 53774;
53775; 10494; 10495; 53776; 12363; 6586; 53777; 53778; 53779;
53780; 53781; 53782; 53783; 53784; 53785; 53786; 53787; 53788;
53789; 53790; 53791; 53792; 53793; 53794; 29286; 13044; 53795; 668;
53796; 53797; 53798; 23890; 4584; 13047; 6608; 53799; 53800; 53801;
53802; 53803; 20399; 20400; 3270; 53804; 53805; 53806; 53807;
29302; 53808; 6615; 25594; 53809; 10619; 14810; 53810; 53811; 4608;
4609; 4610; 53812; 53813; 53814; 53815; 53816; 53817; 53818; 53819;
53820; 53821; 53822; 6629; 53823; 3283; 4615; 6630; 10629; 53824;
53825; 53826; 53827; 4618; 4619; 4620; 53828; 7839; 53829; 53830;
53831; 19710; 7845; 3297; 12398; 53832; 4630; 53833; 53834; 3307;
53835; 6646; 6647; 6648; 6649; 6650; 6651; 6652; 6653; 53836;
53837; 1577; 4641; 53838; 22575; 53839; 6659; 3314; 6661; 3315;
6660; 53840; 53841; 53842; 53843; 53844; 6718; 53845; 3329.
[0227] The following SEQ ID NOs correspond to the polynucleotides
encoding breast-specific proteins as described in Table 51A
identified using SBS: 17025; 14099; 8321; 54808; 54809; 17063;
17064; 17080; 54810; 54811; 54812; 54813; 54814; 54815; 54816;
54817; 54818; 54819; 54820; 54821; 17132; 54822; 17152; 17153;
24429; 17171; 17172.
[0228] The following SEQ ID NOs correspond to the amino acid
sequences of breast-specific proteins as described in Table 51A
identified using SBS: 17185; 14513; 9649; 54823; 54824; 17223;
17224; 17240; 54825; 54826; 54827; 54828; 54829; 54830; 54831;
54832; 54833; 54834; 54835; 54836; 17292; 54837; 17312; 17313;
24559; 17331; 17332.
[0229] The following SEQ ID NOs correspond to the polynucleotides
encoding cervix-specific proteins as described in Table 52A
identified using SBS: 14134; 32022; 54868; 14256; 54869; 54870;
54871; 54872; 54873; 54874.
[0230] The following SEQ ID NOs correspond to the amino acid
sequences of cervix-specific proteins as described in Table 52A
identified using SBS: 14548; 32085; 54875; 14670; 54876; 54877;
54878; 54879; 54880; 54881.
[0231] The following SEQ ID NOs correspond to the polynucleotides
encoding heart-specific proteins as described in Table 53A
identified using SBS: 1030; 14040; 8132; 31350; 54896; 14053;
54897; 54898; 14076; 3660; 3661; 3662; 3663; 3664; 3665; 3666;
3667; 14083; 14098; 54899; 14103; 14107; 20608; 14117; 54900;
54901; 14144; 14159; 14160; 27769; 54902; 54903; 14200; 54904;
54905; 54906; 54907; 3808; 3809; 3810; 3811; 3812; 3813; 3814;
14227; 14241; 14244; 14247; 54908; 14254; 14273; 14274; 14275;
14277; 14278; 54909; 54910; 14280; 14282; 14287; 14288; 14292;
14293; 14294; 14295; 14296; 54911; 54912; 54913; 54914; 14332;
54915; 54916; 54917; 54918; 54919; 54920; 54921; 14347; 54922;
14363; 54923; 54924; 14373; 14378; 54925; 14383; 14388; 54926;
54927; 54928; 54929; 14400; 20263; 14411; 14412; 14413; 14414;
14415; 14416; 1285; 1286; 1287; 1288; 1289; 1290; 1291; 14423;
14424; 14425; 14426; 54930; 2952; 2953; 2955; 2956; 14433;
14434.
[0232] The following SEQ ID NOs correspond to the amino acid
sequences of heart-specific proteins as described in Table 53A
identified using SBS: 1313; 14454; 9460; 31512; 54931; 14467;
54932; 54933; 14490; 4209; 4210; 4211; 4212; 4213; 4214; 4215;
4216; 14497; 14512; 54934; 14517; 14521; 20875; 14531; 54935;
54936; 14558; 14573; 14574; 28771; 54937; 54938; 14614; 54939;
54940; 54941; 54942; 4357; 4358; 4359; 4360; 4361; 4362; 4363;
14641; 14658; 14655; 14661; 54943; 14668; 14687; 14688; 14689;
14691; 14692; 54944; 54945; 14694; 14696; 14701; 14702; 14706;
14707; 14708; 14709; 14710; 54946; 54947; 54948; 54949; 14746;
54950; 54951; 54952; 54953; 54954; 54955; 54956; 14761; 54957;
14777; 54958; 54959; 14787; 14792; 54960; 14797; 14802; 54961;
54962; 54963; 54964; 14814; 20404; 14825; 14826; 14827; 14828;
14829; 14830; 1568; 1569; 1570; 1571; 1572; 1573; 1574; 14837;
14838; 14839; 14840; 54965; 3308; 3309; 3311; 3312; 14847;
14848.
[0233] The following SEQ ID NOs correspond to the polynucleotides
encoding kidney-specific proteins as described in Table 54A
identified using SBS: 12671; 12672; 55189; 32757; 32758; 15394;
55190; 32378; 55191; 55192; 55193; 55194; 55195; 55196; 8207;
55197; 55198; 55199; 55200; 15932; 5158; 25869; 30482; 15412;
55201; 55202; 15413; 23310; 8294; 14094; 55203; 55204; 55205;
55206; 55207; 55208; 55209; 55210; 25369; 11972; 20614; 31380;
55211; 5216; 5217; 8403; 55212; 55213; 55214; 55215; 3742; 55216;
55217; 55218; 24367; 24368; 15448; 55219; 55220; 55221; 55222;
55223; 55224; 55225; 55226; 55227; 1943; 55228; 55229; 55230;
55231; 55232; 55233; 55234; 55235; 55236; 55237; 55238; 55239;
55240; 32778; 32779; 27857; 17098; 17099; 17100; 17101; 24815;
55241; 55242; 20695; 55243; 55244; 55245; 55246; 15474; 15473;
15475; 55247; 55248; 55249; 55250; 55251; 55252; 55253; 55254;
55255; 55256; 55257; 55258; 55259; 55260; 55261; 55262; 55263;
55264; 55265; 55266; 55267; 55268; 55269; 55270; 55271; 55272;
55273; 55274; 55275; 55276; 55277; 55278; 55279; 55280; 15479;
55281; 55282; 55283; 55284; 55285; 55286; 55287; 55288; 55289;
55290; 55291; 3898; 5465; 55292; 8893; 32436; 55293; 8908; 55294;
15494; 55295; 55296; 55297; 55298; 8963; 8964; 15498; 15499; 15500;
15501; 15502; 31450; 31451; 31452; 31453; 31454; 23486; 15504;
15507; 55299; 55300; 55301; 55302; 55303; 16183; 16184; 55304;
55305; 55306; 55307; 15522; 15523; 15524; 55308; 15525; 15526;
5766; 5767; 5770; 55309; 55310; 55311; 55312; 15529; 2915; 55313;
55314; 12844; 2936; 55315; 55316; 20816; 2073; 55317; 55318; 55319;
55320; 30707; 30708; 55321; 15547; 15548; 55322; 55323; 55324;
28424.
[0234] The following SEQ ID NOs correspond to the amino acid
sequences of kidney-specific proteins as described in Table 54A
identified using SBS: 12879; 12880; 55325; 32813; 32814; 15570;
55326; 32485; 55327; 55328; 55329; 55330; 55331; 55332; 9535;
55333; 55334; 55335; 55336; 16329; 5992; 26339; 30745; 15588;
55337; 55338; 15589; 23641; 9622; 14508; 55339; 55340; 55341;
55342; 55343; 55344; 55345; 55346; 25509; 12228; 20881; 31542;
55347; 6050; 6051; 9731; 55348; 55349; 55350; 55351; 4291; 55352;
55353; 55354; 24497; 24498; 15624; 55355; 55356; 55357; 55358;
55359; 55360; 55361; 55362; 55363; 2242; 55364; 55365; 55366;
55367; 55368; 55369; 55370; 55371; 55372; 55373; 55374; 55375;
55376; 32834; 32835; 28859; 17261; 17259; 17260; 17258; 25018;
55377; 55378; 20962; 55379; 55380; 55381; 55382; 15649; 15650;
15651; 55383; 55384; 55385; 55386; 55387; 55388; 55389; 55390;
55391; 55392; 55393; 55394; 55395; 55396; 55397; 55398; 55399;
55400; 55401; 55402; 55403; 55404; 55405; 55406; 55407; 55408;
55409; 55410; 55411; 55412; 55413; 55414; 55415; 55416; 15655;
55417; 55418; 55419; 55420; 55421; 55422; 55423; 55424; 55425;
55426; 55427; 4447; 6299; 55428; 10221; 32543; 55429; 10236; 55430;
15670; 55431; 55432; 55433; 55434; 10291; 10292; 15674; 15675;
15676; 15677; 15678; 31612; 31613; 31614; 31615; 31616; 23817;
15680; 15683; 55435; 55436; 55437; 55438; 55439; 16580; 16581;
55440; 55441; 55442; 55443; 15698; 15699; 15700; 55444; 15701;
15702; 6600; 6601; 6604; 55445; 55446; 55447; 55448; 15705; 3271;
55449; 55450; 13052; 3292; 55451; 55452; 21083; 2372; 55453; 55454;
55455; 55456; 30970; 30971; 55457; 15724; 15723; 55458; 55459;
55460; 29426.
[0235] The following SEQ ID NOs correspond to the polynucleotides
encoding liver-specific proteins as described in Table 55A
identified using SBS: 55676; 55677; 55678; 55679; 55680; 55681;
55682; 55683; 24715; 11906; 55684; 55685; 55686; 55687; 15375;
55688; 15377; 55689; 55690; 55691; 55692; 5088; 1036; 55693; 55694;
55695; 15383; 55696; 20139; 17507; 17508; 55697; 32311; 8157;
23281; 15389; 15390; 15391; 55698; 55699; 55700; 15393; 17; 20585;
55701; 55702; 55703; 3598; 18; 55704; 8172; 55705; 55706; 55707;
1814; 55708; 55709; 55710; 55711; 55712; 23; 55713; 55714; 55715;
55716; 55717; 55718; 55719; 5142; 55720; 55721; 55722; 55723;
55724; 55725; 55726; 55727; 55728; 55729; 55730; 55731; 55732;
31367; 31368; 55733; 55734; 55735; 55736; 55737; 19014; 55738; 55;
55739; 55740; 55741; 23315; 23316; 55742; 55743; 19016; 55744;
55745; 55746; 55747; 55748; 55749; 55750; 23326; 55751; 55752;
24330; 24331; 15425; 15426; 55753; 55754; 55755; 55756; 55757;
55758; 55759; 55760; 55761; 55762; 55763; 55764; 24774; 55765;
55766; 3698; 55767; 24775; 24776; 24777; 24778; 55768; 15427;
16001; 24779; 24780; 55769; 55770; 1878; 55771; 55772; 55773;
55774; 55775; 55776; 25375; 20620; 1886; 55777; 13344; 55778;
55779; 55780; 55781; 25380; 23357; 55782; 13345; 55783; 55784;
55785; 55786; 55787; 55788; 55789; 55790; 55791; 55792; 55793;
55794; 55795; 55796; 55797; 55798; 55799; 55800; 55801; 15446;
15447; 55802; 55803; 55804; 14167; 55805; 55806; 55807; 55808;
55809; 55810; 15450; 55811; 13365; 55812; 55813; 55814; 55815;
55816; 55817; 11999; 12000; 55818; 55819; 19105; 14184; 14185;
14186; 14187; 55820; 55821; 55822; 55823; 55824; 55825; 55826;
55827; 14198; 55828; 55829; 15453; 55830; 55831; 55832; 55833;
55834; 55835; 55836; 55837; 55838; 20675; 55839; 55840; 55841;
55842; 55843; 55844; 55845; 55846; 15469; 55847; 55848; 3851;
55849; 55850; 8732; 15472; 55851; 55852; 55853; 55854; 27910;
27909; 55855; 55856; 55857; 55858; 55859; 55860; 55861; 55862;
55863; 55864; 55865; 55866; 55867; 55868; 55869; 55870; 55871;
55872; 55873; 55874; 55875; 55876; 17111; 55877; 55878; 55879;
55880; 55881; 55882; 55883; 55884; 55885; 55886; 55887; 55888;
55889; 55890; 55891; 55892; 55893; 55894; 55895; 55896; 55897;
55898; 55899; 55900; 55901; 55902; 55903; 55904; 14255; 55905;
55906; 55907; 55908; 7396; 55909; 1975; 1978; 1979; 1980; 55910;
55911; 55912; 55913; 55914; 55915; 26100; 26101; 26102; 55916;
12806; 12807; 55917; 55918; 26108; 55919; 55920; 55921; 55922;
55923; 15503; 20763; 55924; 55925; 15506; 55926; 55927; 55928;
55929; 55930; 55931; 55932; 55933; 55934; 55935; 15516; 55936;
55937; 55938; 55939; 55940; 55941; 55942; 55943; 55944; 55945;
55946; 55947; 55948; 2030; 55949; 55950; 17152; 17153; 55951;
55952; 55953; 13475; 13476; 13477; 55954; 55955; 55956; 55957;
55958; 55959; 55960; 55961; 55962; 55963; 9189; 55964; 55965;
55966; 55967; 55968; 55969; 55970; 55971; 55972; 55973; 55974;
55975; 55976; 21924; 15527; 7478; 21926; 14377; 55977; 32460;
55978; 55979; 319; 17164; 55980; 7514; 2072; 55981; 55982; 55983;
55984; 12863; 55985; 55986; 55987; 55988; 55989; 55990; 55991;
55992; 55993; 55994; 15545; 55995; 27358; 13526; 55996; 14431;
15546; 26236; 15549; 55997; 28388.
[0236] The following SEQ ID NOs correspond to the amino acid
sequences of liver-specific proteins as described in Table 55A
identified using SBS: 55998; 55999; 56000; 56001; 56002; 56003;
56004; 56005; 24918; 12162; 56006; 56007; 56008; 56009; 15551;
56010; 15553; 56011; 56012; 56013; 56014; 5922; 1319; 56015; 56016;
56017; 15559; 56018; 20280; 17976; 17977; 56019; 32329; 9485;
23612; 15565; 15566; 15567; 56020; 56021; 56022; 15569; 385; 20852;
56023; 56024; 56025; 4147; 386; 56026; 9500; 56027; 56028; 56029;
2113; 56030; 56031; 56032; 56033; 56034; 391; 56035; 56036; 56037;
56038; 56039; 56040; 56041; 5976; 56042; 56043; 56044; 56045;
56046; 56047; 56048; 56049; 56050; 56051; 56052; 56053; 56054;
31530; 31529; 56055; 56056; 56057; 56058; 56059; 19418; 56060; 423;
56061; 56062; 56063; 23646; 23647; 56064; 56065; 19420; 56066;
56067; 56068; 56069; 56070; 56071; 56072; 23657; 56073; 56074;
24460; 24461; 15601; 15602; 56075; 56076; 56077; 56078; 56079;
56080; 56081; 56082; 56083; 56084; 56085; 56086; 24977; 56087;
56088; 4247; 56089; 24978; 24979; 24980; 24981; 56090; 15603;
16398; 24982; 24983; 56091; 56092; 2177; 56093; 56094; 56095;
56096; 56097; 56098; 25515; 20887; 2185; 56099; 13611; 56100;
56101; 56102; 56103; 25520; 23688; 56104; 13612; 56105; 56106;
56107; 56108; 56109; 56110; 56111; 56112; 56113; 56114; 56115;
56116; 56117; 56118; 56119; 56120; 56121; 56122; 56123; 15622;
15623; 56124; 56125; 56126; 14581; 56127; 56128; 56129; 56130;
56131; 56132; 15626; 56133; 13632; 56134; 56135; 56136; 56137;
56138; 56139; 12256; 12255; 56140; 56141; 19509; 14598; 14599;
14600; 14601; 56142; 56143; 56144; 56145; 56146; 56147; 56148;
56149; 14612; 56150; 56151; 15629; 56152; 56153; 56154; 56155;
56156; 56157; 56158; 56159; 56160; 20942; 56161; 56162; 56163;
56164; 56165; 56166; 56167; 56168; 15645; 56169; 56170; 4400;
56171; 56172; 10060; 15648; 56173; 56174; 56175; 56176; 28911;
28912; 56177; 56178; 56179; 56180; 56181; 56182; 56183; 56184;
56185; 56186; 56187; 56188; 56189; 56190; 56191; 56192; 56193;
56194; 56195; 56196; 56197; 56198; 17271; 56199; 56200; 56201;
56202; 56203; 56204; 56205; 56206; 56207; 56208; 56209; 56210;
56211; 56212; 56213; 56214; 56215; 56216; 56217; 56218; 56219;
56220; 56221; 56222; 56223; 56224; 56225; 56226; 14669; 56227;
56228; 56229; 56230; 7726; 56231; 2274; 2277; 2278; 2279; 56232;
56233; 56234; 56235; 56236; 56237; 26570; 26571; 26572; 56238;
13014; 13015; 56239; 56240; 26578; 56241; 56242; 56243; 56244;
56245; 15679; 21030; 56246; 56247; 15682; 56248; 56249; 56250;
56251; 56252; 56253; 56254; 56255; 56256; 56257; 15692; 56258;
56259; 56260; 56261; 56262; 56263; 56264; 56265; 56266; 56267;
56268; 56269; 56270; 2329; 56271; 56272; 17312; 17313; 56273;
56274; 56275; 13743; 13744; 13742; 56276; 56277; 56278; 56279;
56280; 56281; 56282; 56283; 56284; 56285; 10517; 56286; 56287;
56288; 56289; 56290; 56291; 56292; 56293; 56294; 56295; 56296;
56297; 56298; 22511; 15703; 7808; 22513; 14791; 56299; 32567;
56300; 56301; 687; 17324; 56302; 7844; 2371; 56303; 56304; 56305;
56306; 13071; 56307; 56308; 56309; 56310; 56311; 56312; 56313;
56314; 56315; 56316; 15721; 56317; 27388; 13793; 56318; 14845;
15722; 26706; 15725; 56319; 29390.
[0237] The following SEQ ID NOs correspond to the polynucleotides
encoding lung-specific proteins as described in Table 56A
identified using SBS: 57163; 57164; 8146; 8147; 57165; 25344;
25345; 8190; 57166; 57167; 57168; 57169; 57170; 30481; 57171;
13324; 15987; 15988; 57172; 16015; 16016; 57173; 57174; 57175;
57176; 57177; 57178; 57179; 57180; 57181; 57182; 57183; 57184;
57185; 57186; 57187; 57188; 21631; 16039; 7343; 13373; 57189; 1940;
1941; 16054; 57190; 16062; 57191; 57192; 57193; 57194; 57195;
57196; 57197; 57198; 57199; 57200; 57201; 57202; 57203; 57204;
16079; 16080; 16081; 16082; 16083; 16084; 57205; 57206; 57207;
57208; 57209; 57210; 57211; 57212; 16089; 16090; 16091; 16092;
16093; 16094; 16095; 57213; 31429; 57214; 16121; 57215; 57216;
1996; 16147; 57217; 57218; 57219; 57220; 26177; 57221; 16187;
57222; 16191; 57223; 16192; 16193; 16194; 16195; 57224; 13506;
2928; 2929; 57225; 57226; 20835,
[0238] The following SEQ ID NOs correspond to the amino acid
sequences of lung-specific proteins as described in Table 56A
identified using SBS: 57227; 57228; 9474; 9475; 57229; 25484;
25485; 9518; 57230; 57231; 57232; 57233; 57234; 30744; 57235;
13591; 16384; 16385; 57236; 16412; 16413; 57237; 57238; 57239;
57240; 57241; 57242; 57243; 57244; 57245; 57246; 57247; 57248;
57249; 57250; 57251; 57252; 22218; 16436; 7673; 13640; 57253; 2239;
2240; 16451; 57254; 16459; 57255; 57256; 57257; 57258; 57259;
57260; 57261; 57262; 57263; 57264; 57265; 57266; 57267; 57268;
16480; 16479; 16477; 16478; 16476; 16481; 57269; 57270; 57271;
57272; 57273; 57274; 57275; 57276; 16486; 16487; 16488; 57277;
16489; 16491; 16492; 57278; 31591; 57279; 16518; 57280; 57281;
2295; 16544; 57282; 57283; 57284; 57285; 26647; 57286; 16584;
16490; 16588; 57287; 16589; 16590; 16591; 16592; 57288; 13773;
3284; 3285; 57289; 57290; 21102.
[0239] The following SEQ ID NOs correspond to the polynucleotides
encoding lymph node-specific proteins as described in Table 57A
identified using SBS: 57417; 57418; 57419; 30488; 57420; 57421;
57422; 57423; 57424; 57425; 57426; 25893; 25894; 57427; 57428;
57429; 57430; 57431; 25403; 57432; 57433; 1951; 1956; 57434; 26033;
1957; 1959; 26034; 1961; 57435; 26035; 57436; 57437; 57438; 1963;
1967; 1968; 57439; 57440; 57441; 57442; 26132; 57443; 57444; 57445;
57446; 57447; 19327.
[0240] The following SEQ ID NOs correspond to the amino acid
sequences of lymph node-specific proteins as described in Table 57A
identified using SBS: 57448; 57449; 57450; 30751; 57451; 57452;
57453; 57454; 57455; 57456; 57457; 26363; 26364; 57458; 57459;
57460; 57461; 57462; 25543; 57463; 57464; 2250; 2255; 57465; 26503;
2256; 2258; 26504; 2260; 57466; 26505; 57467; 57468; 57469; 2262;
2266; 2267; 57470; 57471; 57472; 57473; 26602; 57474; 57475; 57476;
57477; 57478; 19731.
[0241] The following SEQ ID NOs correspond to the polynucleotides
encoding lymphocyte-specific proteins as described in Table 58A
identified using SBS: 57517; 8200; 24728; 30473; 30474; 8214;
57518; 24733; 25844; 25845; 57519; 57520; 57521; 57522; 57523;
57524; 32389; 57525; 57526; 19007; 11956; 57527; 57528; 30488;
57420; 57529; 57530; 57531; 57532; 25881; 25882; 57533; 57534;
57535; 57536; 57537; 57538; 57539; 57540; 57541; 57542; 57543;
57544; 1874; 1875; 57545; 57546; 57547; 57548; 57549; 57550; 57551;
25935; 25936; 57552; 57553; 57554; 57555; 57556; 25949; 57557;
19098; 19099; 57558; 24371; 57559; 19102; 19103; 57560; 57561;
57562; 57563; 57564; 57565; 57566; 57567; 25978; 57568; 16056;
8656; 30541; 57569; 8715; 57570; 57571; 57572; 57573; 57574; 57575;
57576; 57577; 57578; 57579; 57580; 57581; 19127; 27865; 25407;
57582; 57583; 57584; 57585; 57586; 57587; 57588; 57589; 8803;
57590; 57591; 8793; 57592; 57593; 57594; 57595; 57596; 57597;
57598; 57599; 57600; 57601; 57602; 57603; 57604; 57605; 57606;
57607; 26075; 26076; 57608; 57609; 57610; 57611; 57612; 26112;
26113; 19197; 57613; 57614; 7419; 57615; 5707; 5708; 5709; 5710;
5711; 30668; 57616; 57617; 57618; 57619; 57620; 30685; 30686; 5783;
57621; 57622; 57623; 57624; 2941; 57625; 57626; 57627; 57628;
57629; 57630; 57631; 57632; 57633; 57634; 30728; 7540.
[0242] The following SEQ ID NOs correspond to the amino acid
sequences of lymphocyte-specific proteins as described in Table 58A
identified using SBS: 57635; 9528; 24931; 30736; 30737; 9542;
57636; 24936; 26314; 26315; 57637; 57638; 57639; 57640; 57641;
57642; 32496; 57643; 57644; 19411; 12212; 57645; 57646; 30751;
57451; 57647; 57648; 57649; 57650; 26351; 26352; 57651; 57652;
57653; 57654; 57655; 57656; 57657; 57658; 57659; 57660; 57661;
57662; 2173; 2174; 57663; 57664; 57665; 57666; 57667; 57668; 57669;
26405; 26406; 57670; 57671; 57672; 57673; 57674; 26419; 57675;
19502; 19503; 57676; 24501; 57677; 19506; 19507; 57678; 57679;
57680; 57681; 57682; 57683; 57684; 57685; 26448; 57686; 16453;
9984; 30804; 57687; 10043; 57688; 57689; 57690; 57691; 57692;
57693; 57694; 57695; 57696; 57697; 57698; 57699; 19531; 28867;
25547; 57700; 57701; 57702; 57703; 57704; 57705; 57706; 57707;
10131; 57708; 57709; 10121; 57710; 57711; 57712; 57713; 57714;
57715; 57716; 57717; 57718; 57719; 57720; 57721; 57722; 57723;
57724; 57725; 26546; 26545; 57726; 57727; 57728; 57729; 57730;
26583; 26582; 19601; 57731; 57732; 7749; 57733; 6541; 6542; 6543;
6544; 6545; 30931; 57734; 57735; 57736; 57737; 57738; 30948; 30949;
6617; 57739; 57740; 57741; 57742; 3297; 57743; 57744; 57745; 57746;
57747; 57748; 57749; 57750; 57751; 57752; 30991; 7870.
[0243] The following SEQ ID NOs correspond to the polynucleotides
encoding monocyte-specific proteins as described in Table 59A
identified using SBS: 57886; 57887; 57888; 18969; 18970; 18971;
18972; 18973; 57889; 57890; 17536; 17548; 57891; 57892; 57893;
57894; 57533; 25886; 57895; 57896; 1857; 57897; 17569; 17570;
17571; 17572; 57898; 1086; 57899; 17613; 1900; 1901; 1902; 1903;
1904; 1905; 57900; 1906; 114; 21617; 54868; 57901; 17675; 17676;
17683; 14207; 8661; 30547; 19131; 57902; 17706; 57903; 57904;
57905; 57906; 57907; 57908; 57909; 57910; 57911; 57912; 57913;
57914; 57915; 57916; 57917; 57918; 57919; 57920; 57921; 57922;
57923; 1972; 17792; 17799; 17800; 17801; 17802; 17803; 17804;
23466; 57924; 8966; 16140; 16144; 19204; 7426; 7427; 2007; 19208;
57925; 57926; 57927; 57928; 57929; 20253; 9231; 57930; 57931;
26224; 19316; 57932.
[0244] The following SEQ ID NOs correspond to the amino acid
sequences of monocyte-specific proteins as described in Table 59A
identified using SBS: 57933; 57934; 57935; 19373; 19374; 19375;
19376; 19377; 57936; 57937; 18005; 18017; 57938; 57939; 57940;
57941; 57651; 26356; 57942; 57943; 2156; 57944; 18038; 18039;
18040; 18041; 57945; 1369; 57946; 18082; 2199; 2200; 2201; 2202;
2203; 2204; 57947; 2205; 482; 22204; 54875; 57948; 18144; 18145;
18152; 14621; 9989; 30810; 19535; 57949; 18175; 57950; 57951;
57952; 57953; 57954; 57955; 57956; 57957; 57958; 57959; 57960;
57961; 57962; 57963; 57964; 57965; 57966; 57967; 57968; 57969;
57970; 2271; 18261; 18270; 18269; 18272; 18271; 18273; 18268;
23797; 57971; 10294; 16537; 16541; 19608; 7756; 7757; 2306; 19612;
57972; 57973; 57974; 57975; 57976; 20394; 10559; 57977; 57978;
26694; 19720; 57979.
[0245] The following SEQ ID NOs correspond to the polynucleotides
encoding muscle-specific proteins as described in Table 60A
identified using SBS: 1796; 1797; 1798; 1030; 8132; 31350; 27432;
27433; 58069; 58070; 58071; 58072; 58073; 58074; 58075; 14044;
14045; 14046; 14047; 14048; 14049; 14050; 14051; 58076; 58077;
58078; 3615; 3617; 8192; 8193; 8194; 8195; 3633; 58079; 58080;
58081; 58082; 58083; 58084; 58085; 21514; 58086; 58087; 58088;
58089; 58090; 58091; 58092; 58093; 58094; 14084; 14085; 27605;
58095; 58096; 14097; 58097; 14102; 14103; 14108; 58098; 53114;
58099; 58100; 58101; 27664; 27665; 27666; 27667; 27668; 27669;
3719; 58102; 1105; 1106; 14141; 14142; 58103; 58104; 58105; 125;
58106; 14184; 14185; 14186; 14187; 58107; 58108; 25984; 58109;
58110; 58111; 58112; 14227; 14230; 58113; 58114; 58115; 58116;
58117; 58118; 58119; 14233; 58120; 58121; 58122; 58123; 58124;
58125; 14235; 58126; 58127; 58128; 58129; 58130; 58131; 58132;
58133; 58134; 58135; 58136; 58137; 58138; 14245; 58139; 58140;
58141; 58142; 58143; 58144; 58145; 14248; 58146; 58147; 58148;
14257; 14258; 14259; 8871; 21800; 21801; 21802; 21803; 58149;
58150; 58151; 31436; 8910; 31437; 31438; 31439; 14275; 31440;
58152; 58153; 58154; 1983; 14279; 58155; 58156; 14281; 58157;
58158; 14283; 2844; 14295; 14296; 58159; 58160; 58161; 58162;
58163; 58164; 58165; 14312; 14314; 14315; 58166; 14317; 14318;
14319; 14320; 14321; 17847; 58167; 52997; 58168; 58169; 32802;
14337; 58170; 58171; 2018; 58172; 2895; 58173; 14358; 58174; 58175;
58176; 58177; 58178; 58179; 1250; 1251; 58180; 2907; 58181; 58182;
58183; 14383; 58184; 9278; 54926; 54927; 54928; 54929; 58185;
58186; 58187; 58188; 58189; 2070; 58190; 58191; 14410; 58192;
58193; 58194; 58195; 58196; 1292; 1293; 17933; 17934; 14421; 58197;
58198; 2952; 2953; 2954; 2955; 2956; 58199; 58200; 1307.
[0246] The following SEQ ID NOs correspond to the amino acid
sequences of muscle-specific proteins as described in Table 60A
identified using SBS: 2095; 2096; 2097; 1313; 9460; 31512; 28434;
28435; 58201; 58202; 58203; 58204; 58205; 58206; 58207; 14458;
14459; 14460; 14461; 14462; 14463; 14464; 14465; 58208; 58209;
58210; 4164; 4166; 9520; 9521; 9522; 9523; 4182; 58211; 58212;
58213; 58214; 58215; 58216; 58217; 22101; 58218; 58219; 58220;
58221; 58222; 58223; 58224; 58225; 58226; 14499; 14498; 28607;
58227; 58228; 14511; 58229; 14516; 14517; 14522; 58230; 53519;
58231; 58232; 58233; 28666; 28667; 28668; 28669; 28670; 28671;
4268; 58234; 1388; 1389; 14556; 14555; 58235; 58236; 58237; 493;
58238; 14598; 14599; 14600; 14601; 58239; 58240; 26454; 58241;
58242; 58243; 58244; 14641; 14644; 58245; 58246; 58247; 58248;
58249; 58250; 58251; 14647; 58252; 58253; 58254; 58255; 58256;
58257; 58258; 14649; 58259; 58260; 58261; 58262; 58263; 58264;
58265; 58266; 58267; 58268; 58269; 58270; 14659; 58271; 58272;
58273; 58274; 58275; 58276; 58277; 14662; 58278; 58279; 58280;
14673; 14671; 14672; 10199; 22387; 22388; 22389; 22390; 58281;
58282; 58283; 31598; 10238; 31599; 31600; 31601; 14689; 31602;
58284; 58285; 58286; 2282; 14693; 58287; 58288; 14695; 58289;
58290; 14697; 3200; 14709; 14710; 58291; 58292; 58293; 58294;
58295; 58296; 58297; 14726; 14728; 14729; 58298; 14731; 14732;
14733; 14734; 14735; 18316; 58299; 53012; 58300; 58301; 32858;
14751; 58302; 58303; 2317; 58304; 3251; 58305; 14772; 58306; 58307;
58308; 58309; 58310; 58311; 1533; 1534; 58312; 3263; 58313; 58314;
58315; 14797; 58316; 10606; 54961; 54962; 54963; 54964; 58317;
58318; 58319; 58320; 58321; 2369; 58322; 58323; 14824; 58324;
58325; 58326; 58327; 58328; 1575; 1576; 18402; 18403; 14835; 58329;
58330; 3308; 3309; 3310; 3311; 3312; 58331; 58332; 1590.
[0247] The following SEQ ID NOs correspond to the polynucleotides
encoding ovary-specific proteins as described in Table 61A
identified using SBS: 58733; 58734; 58735; 58736; 58737; 58738;
58739; 58740; 58741; 58742; 32270; 28222; 58743; 58744; 58745;
58746.
[0248] The following SEQ ID NOs correspond to the amino acid
sequences of ovary-specific proteins as described in Table 61A
identified using SBS: 58747; 58748; 58749; 58750; 58751; 58752;
58753; 58754; 58755; 58756; 32284; 29224; 58757; 58758; 58759;
58760.
[0249] The following SEQ ID NOs correspond to the polynucleotides
encoding pancreas-specific proteins as described in Table 62A
identified using SBS: 5071; 58778; 18844; 18845; 18846; 18847;
18848; 17509; 13275; 58779; 58780; 58781; 58782; 58783; 58784;
58785; 58786; 58787; 58788; 5177; 58789; 58790; 18850; 18851;
18852; 18853; 18854; 8338; 18855; 58791; 21555; 58792; 18856;
58793; 18857; 58794; 105; 58795; 25929; 58796; 58797; 8530; 58798;
2725; 8554; 58799; 58800; 58801; 58802; 58803; 23389; 58804; 58805;
58806; 58807; 58808; 58809; 58810; 58811; 58812; 13387; 58813;
58814; 58815; 58816; 58817; 58818; 58819; 58820; 58821; 55265;
55267; 18858; 18859; 58822; 58823; 18860; 58824; 58825; 58826;
58827; 58828; 58829; 58830; 58831; 58832; 58833; 58834; 5629;
58835; 24865; 8978; 8979; 58836; 20245; 58837; 18862; 58838; 58839;
18863; 18864; 18865; 13444; 13445; 13446; 13447; 13448; 13449;
13450; 58840; 18866; 18867; 58841; 58842; 18869; 18870; 18871;
24874; 24875; 24876; 24877; 24878; 58843; 20254; 58844; 58845;
58846; 18872; 58847; 58848; 58849; 58850; 23559; 1257; 58851;
58852; 5811; 58853; 58854; 58855; 58856; 58857; 58858; 7540.
[0250] The following SEQ ID NOs correspond to the amino acid
sequences of pancreas-specific proteins as described in Table 62A
identified using SBS: 5905; 58859; 18875; 18874; 18876; 18873;
18877; 17978; 13542; 58860; 58861; 58862; 58863; 58864; 58865;
58866; 58867; 58868; 58869; 6011; 58870; 58871; 18879; 18880;
18881; 18882; 18883; 9666; 18884; 58872; 22142; 58873; 18885;
58874; 18886; 58875; 473; 58876; 26399; 58877; 58878; 9858; 58879;
3081; 9882; 58880; 58881; 58882; 58883; 58884; 23720; 58885; 58886;
58887; 58888; 58889; 58890; 58891; 58892; 58893; 13654; 58894;
58895; 58896; 58897; 58898; 58899; 58900; 58901; 58902; 55401;
55403; 18887; 18888; 58903; 58904; 18889; 58905; 58906; 58907;
58908; 58909; 58910; 58911; 58912; 58913; 58914; 58915; 6463;
58916; 25068; 10306; 10307; 58917; 20386; 58918; 18891; 58919;
58920; 18892; 18893; 18894; 13711; 13712; 13713; 13714; 13715;
13716; 13717; 58921; 18895; 18896; 58922; 58923; 18898; 18899;
18900; 25078; 25077; 25079; 25081; 25080; 58924; 20395; 58925;
58926; 58927; 18901; 58928; 58929; 58930; 58931; 23890; 1540;
58932; 58933; 6645; 58934; 58935; 58936; 58937; 58938; 58939;
7870.
[0251] The following SEQ ID NOs correspond to the polynucleotides
encoding prostate-specific proteins as described in Table 63A
identified using SBS: 13270; 21443; 3588; 21444; 21445; 17027;
21460; 59267; 3616; 3618; 8196; 15403; 59268; 21492; 21493; 21494;
21495; 21496; 59269; 59270; 53089; 1847; 1848; 59271; 59272; 59273;
20170; 8343; 59274; 21557; 21558; 21559; 21560; 21561; 20617;
59275; 23359; 59276; 21618; 16037; 59277; 59278; 59279; 59280;
20661; 59281; 21645; 59282; 59283; 8668; 59284; 8669; 55235; 55236;
55237; 59285; 21665; 21666; 21667; 21668; 21669; 21670; 21672;
59286; 1159; 1160; 5391; 17103; 5392; 59287; 59288; 59289; 59290;
59291; 59292; 59293; 20702; 59294; 24389; 59295; 59296; 59297;
59298; 59299; 59300; 21715; 21717; 59301; 59302; 59303; 21723;
21724; 21725; 21726; 21727; 21728; 21729; 21730; 21731; 21732;
21733; 21734; 21735; 21736; 21737; 59304; 59305; 59306; 59307;
21742; 59308; 59309; 59310; 21786; 21787; 59311; 59312; 59313;
21800; 21801; 21802; 21803; 59314; 59315; 21821; 59316; 21829;
20751; 21838; 59317; 59318; 59319; 59320; 59321; 59322; 21853;
21854; 21855; 21856; 21857; 21858; 21859; 32804; 267; 32325; 32326;
59323; 59324; 59325; 32216; 59326; 59327; 32455; 30675; 59328;
21912; 21913; 59329; 59330; 59331; 59332; 12121; 59333; 21932;
9273; 57623; 57624; 21960; 59334; 21961; 21972; 59335; 21980;
59336; 32063; 59337; 59338; 59339; 22001; 59340.
[0252] The following SEQ ID NOs correspond to the amino acid
sequences of prostate-specific proteins as described in Table 63A
identified using SBS: 13537; 22030; 4137; 22031; 22032; 17187;
22047; 59341; 4165; 4167; 9524; 15579; 59342; 22079; 22080; 22082;
22081; 22083; 59343; 59344; 53494; 2147; 2146; 59345; 59346; 59347;
20311; 9671; 59348; 22144; 22145; 22146; 22147; 22148; 20884;
59349; 23690; 59350; 22205; 16434; 59351; 59352; 59353; 59354;
20928; 59355; 22232; 59356; 59357; 9996; 59358; 9997; 55371; 55372;
55373; 59359; 22252; 22253; 22254; 22255; 22256; 22257; 22259;
59360; 1442; 1443; 6225; 17263; 6226; 59361; 59362; 59363; 59364;
59365; 59366; 59367; 20969; 59368; 24519; 59369; 59370; 59371;
59372; 59373; 59374; 22302; 22304; 59375; 59376; 59377; 22310;
22311; 22312; 22313; 22314; 22316; 22315; 22317; 22318; 22319;
22320; 22322; 22321; 22323; 22324; 59378; 59379; 59380; 59381;
22329; 59382; 59383; 59384; 22373; 22374; 59385; 59386; 59387;
22387; 22388; 22389; 22390; 59388; 59389; 22408; 59390; 22416;
21018; 22425; 59391; 59392; 59393; 59394; 59395; 59396; 22440;
22441; 22442; 22443; 22444; 22445; 22446; 32860; 635; 32343; 32344;
59397; 59398; 59399; 32230; 59400; 59401; 32562; 30938; 59402;
22499; 22500; 59403; 59404; 59405; 59406; 12377; 59407; 22519;
10601; 57741; 57742; 22547; 59408; 22548; 22559; 59409; 22567;
59410; 32126; 59411; 59412; 59413; 22588; 59414.
[0253] The following SEQ ID NOs correspond to the polynucleotides
encoding skin-specific proteins as described in Table 64A
identified using SBS: 59601; 59602; 59603; 59604; 59605; 59606;
59607; 59608; 59609; 59610; 32384; 32383; 32385; 59611; 59612;
24318; 24320; 21516; 30483; 59613; 59614; 59615; 59616; 59272;
59617; 59273; 59618; 59619; 59620; 59621; 59622; 59623; 59624;
19055; 59625; 59626; 59627; 59628; 59629; 59630; 97; 98; 2699;
2700; 2701; 2702; 59631; 17615; 59632; 59633; 59634; 59635; 32414;
32415; 59636; 59637; 27794; 27795; 27796; 59638; 59639; 59640;
59641; 59642; 59643; 59644; 59645; 59646; 59647; 59648; 59649;
59283; 59650; 59651; 59652; 59653; 59654; 59655; 59656; 59657;
59658; 59659; 59660; 59661; 59662; 59663; 59664; 59665; 59666;
59667; 59668; 59669; 59670; 59671; 59672; 59673; 59674; 17102;
59675; 59676; 59677; 59678; 59679; 59680; 59681; 59682; 5391;
17103; 5392; 59683; 59684; 59685; 59686; 59687; 59688; 20694;
59689; 59690; 59691; 59692; 59693; 5393; 59694; 59695; 59696;
59697; 59698; 59699; 59700; 30554; 59701; 20696; 59288; 59289;
59290; 59291; 59702; 59703; 59704; 59705; 59706; 59707; 59708;
59709; 59710; 59711; 59712; 59713; 59714; 59715; 59716; 59717;
59718; 59719; 59720; 59721; 59722; 59723; 59724; 59725; 59726;
59727; 59728; 59729; 59730; 59731; 59732; 59733; 59734; 59735;
59736; 59737; 59738; 59739; 59740; 59741; 59742; 59743; 59744;
59745; 59746; 59747; 59748; 59749; 59750; 59751; 59752; 23448;
59753; 59754; 59755; 59756; 59757; 59758; 24398; 23462; 59759;
59760; 59761; 59762; 59763; 59764; 24410; 59765; 59766; 59767;
59768; 59769; 59770; 9046; 24412; 24413; 24414; 24415; 59771;
20775; 59772; 2893; 23522; 59773; 59774; 59775; 30675; 59776;
59777; 59778; 17156; 59779; 17157; 59780; 59781; 17158; 59329;
59782; 59331; 28279; 28280; 23555; 59783; 59784; 59785; 59786;
30697; 59787; 59788; 59789; 59790; 59791; 25457; 25458; 59792;
59793; 23569; 23570; 59794; 59795; 59796; 59797; 1283; 59798;
59799; 59800; 59801; 23585; 23586; 59802; 59803; 59804; 59805;
59806; 59807; 59808; 59809; 59810; 59811; 59812; 59813.
[0254] The following SEQ ID NOs correspond to the amino acid
sequences of skin-specific proteins as described in Table 64A
identified using SBS: 59814; 59815; 59816; 59817; 59818; 59819;
59820; 59821; 59822; 59823; 32491; 32490; 32492; 59824; 59825;
24448; 24450; 22103; 30746; 59826; 59827; 59828; 59829; 59346;
59830; 59347; 59831; 59832; 59833; 59834; 59835; 59836; 59837;
19459; 59838; 59839; 59840; 59841; 59842; 59843; 465; 466; 3055;
3056; 3057; 3058; 59844; 18084; 59845; 59846; 59847; 59848; 32521;
32522; 59849; 59850; 28796; 28797; 28798; 59851; 59852; 59853;
59854; 59855; 59856; 59857; 59858; 59859; 59860; 59861; 59862;
59357; 59863; 59864; 59865; 59866; 59867; 59868; 59869; 59870;
59871; 59872; 59873; 59874; 59875; 59876; 59877; 59878; 59879;
59880; 59881; 59882; 59883; 59884; 59885; 59886; 59887; 17262;
59888; 59889; 59890; 59891; 59892; 59893; 59894; 59895; 6225;
17263; 6226; 59896; 59897; 59898; 59899; 59900; 59901; 20961;
59902; 59903; 59904; 59905; 59906; 6227; 59907; 59908; 59909;
59910; 59911; 59912; 59913; 30817; 59914; 20963; 59362; 59363;
59364; 59365; 59915; 59916; 59917; 59918; 59919; 59920; 59921;
59922; 59923; 59924; 59925; 59926; 59927; 59928; 59929; 59930;
59931; 59932; 59933; 59934; 59935; 59936; 59937; 59938; 59939;
59940; 59941; 59942; 59943; 59944; 59945; 59946; 59947; 59948;
59949; 59950; 59951; 59952; 59953; 59954; 59955; 59956; 59957;
59958; 59959; 59960; 59961; 59962; 59963; 59964; 59965; 23779;
59966; 59967; 59968; 59969; 59970; 59971; 24528; 23793; 59972;
59973; 59974; 59975; 59976; 59977; 24540; 59978; 59979; 59980;
59981; 59982; 59983; 10374; 24542; 24543; 24544; 24545; 59984;
21042; 59985; 3249; 23853; 59986; 59987; 59988; 30938; 59989;
59990; 59991; 17316; 59992; 17317; 59993; 59994; 17318; 59403;
59995; 59405; 29281; 29282; 23886; 59996; 59997; 59998; 59999;
30960; 60000; 60001; 60002; 60003; 60004; 25597; 25598; 60005;
60006; 23900; 23901; 60007; 60008; 60009; 60010; 1566; 60011;
60012; 60013; 60014; 23916; 23917; 60015; 60016; 60017; 60018;
60019; 60020; 60021; 60022; 60023; 60024; 60025; 60026.
[0255] The following SEQ ID NOs correspond to the polynucleotides
encoding small intestine-specific proteins as described in Table
65A identified using SBS: 12670; 60377; 60378; 60379; 24715; 60380;
20576; 60381; 60382; 60383; 60384; 25339; 25340; 25341; 60385;
24717; 60386; 20139; 60387; 15392; 24719; 60388; 60389; 24721;
24722; 60390; 3598; 60391; 60392; 60393; 60394; 60395; 60396;
24727; 60397; 24736; 24737; 15926; 60398; 8231; 24740; 60399;
60400; 60401; 60402; 60403; 60404; 60405; 60406; 60407; 60408;
60409; 60410; 60411; 18981; 31364; 60412; 24760; 60413; 13322;
13323; 60414; 60415; 60416; 3684; 13328; 15420; 60417; 24770;
60418; 1088; 55761; 2693; 60419; 24785; 13336; 24787; 24788; 24789;
60420; 60421; 60422; 5221; 60423; 60424; 60425; 60426; 60427;
60428; 13346; 60429; 60430; 60431; 25386; 25387; 13360; 24799;
24801; 60432; 60433; 8594; 13371; 60434; 13373; 60435; 60436; 152;
24807; 7353; 60437; 60438; 60439; 60440; 15470; 13388; 60441;
15476; 15477; 60442; 60443; 60444; 13395; 13399; 60445; 60446;
60447; 60448; 24848; 60449; 60450; 60451; 60452; 60453; 1958;
24850; 60454; 57436; 60455; 60456; 1969; 60457; 60458; 60459;
55887; 55888; 60460; 60461; 60462; 60463; 60464; 60465; 15480;
60466; 60467; 60468; 60469; 60470; 60471; 13412; 60472; 15483;
60473; 60474; 212; 60475; 60476; 60477; 60478; 13417; 24860; 60479;
60480; 60481; 60482; 60483; 60484; 60485; 24861; 60486; 60487;
60488; 224; 60489; 24865; 13435; 60490; 32444; 60491; 24409; 60492;
13442; 13457; 60493; 60494; 60495; 60496; 13467; 24873; 24879;
60497; 60498; 60499; 60500; 60501; 9172; 24889; 60502; 60503;
60504; 60505; 60506; 60507; 60508; 60509; 13483; 60510; 60511;
60512; 60513; 12120; 17906; 60514; 60515; 60516; 60517; 16201;
60518; 60519; 60520; 16224; 60521; 60522; 24907; 60523; 60524;
13511; 60525; 24908; 13512; 60526; 60527; 60528; 4087; 7517; 13525;
60529; 7527; 21989; 27358; 13526; 7529; 7530; 24912; 24913; 60530;
13530; 60531.
[0256] The following SEQ ID NOs correspond to the amino acid
sequences of small intestine-specific proteins as described in
Table 65A identified using SBS: 12878; 60532; 60533; 60534; 24918;
60535; 20843; 60536; 60537; 60538; 60539; 25479; 25480; 25481;
60540; 24920; 60541; 20280; 60542; 15568; 24922; 60543; 60544;
24924; 24925; 60545; 4147; 60546; 60547; 60548; 60549; 60550;
60551; 24930; 60552; 24939; 24940; 60553; 60554; 9559; 24943;
60555; 60556; 60557; 60558; 60559; 60560; 60561; 60562; 60563;
60564; 60565; 60566; 60567; 19385; 31526; 60568; 24963; 60569;
13589; 13590; 60570; 60571; 60572; 4233; 13595; 15596; 60573;
24973; 60574; 1371; 56083; 3049; 60575; 24988; 13603; 24990; 24991;
24992; 60576; 60577; 60578; 6055; 60579; 60580; 60581; 60582;
60583; 60584; 13613; 60585; 60586; 60587; 25526; 25527; 13627;
25002; 25004; 60588; 60589; 9922; 13638; 60590; 13640; 60591;
60592; 520; 25010; 7683; 60593; 60594; 60595; 60596; 15646; 13655;
60597; 15653; 15652; 60598; 60599; 60600; 13662; 13666; 60601;
60602; 60603; 60604; 25051; 60605; 60606; 60607; 60608; 60609;
2257; 25053; 60610; 57467; 60611; 60612; 2268; 60613; 60614; 16323;
56209; 56210; 60615; 60616; 60617; 60618; 60619; 60620; 15656;
60621; 60622; 60623; 60624; 60625; 60626; 13679; 60627; 15659;
60628; 60629; 580; 60630; 60631; 60632; 60633; 13684; 25063; 60634;
60635; 60636; 60637; 60638; 60639; 60640; 25064; 60641; 60642;
60643; 592; 60644; 25068; 13702; 60645; 32551; 60646; 24539; 60647;
13709; 13724; 60648; 60649; 60650; 60651; 13734; 25076; 25082;
60652; 60653; 60654; 60655; 60656; 10500; 25092; 60657; 60658;
60659; 60660; 60661; 60662; 60663; 60664; 13750; 60665; 60666;
60667; 60668; 12376; 18375; 60669; 60670; 60671; 60672; 16598;
60673; 60674; 60675; 16621; 60676; 60677; 25110; 60678; 60679;
13778; 60680; 25111; 13779; 60681; 60682; 60683; 4636; 7847; 13792;
60684; 7857; 22576; 27388; 13793; 7859; 7860; 25115; 25116; 60685;
13797; 60686.
[0257] The following SEQ ID NOs correspond to the polynucleotides
encoding spleen-specific proteins as described in Table 66A
identified using SBS: 61104; 61105; 61106; 61107; 61108; 61109;
1837; 25880; 61110; 1878; 55771; 61111; 25904; 25927; 61112; 61113;
61114; 25955; 1927; 1928; 1929; 61115; 1937; 25979; 61116; 61117;
61118; 1954; 1953; 61119; 61120; 61121; 61122; 61123; 1960; 1962;
61124; 55891; 23450; 61125; 61126; 61127; 17798; 61128; 61129;
61130; 61131; 61132; 12067; 223; 229; 1995; 61133; 2003; 2032;
61134; 26203; 61135; 61136; 31485; 31486; 31487; 31488; 31489;
61137.
[0258] The following SEQ ID NOs correspond to the amino acid
sequences of spleen-specific proteins as described in Table 66A
identified using SBS: 61138; 61139; 61140; 61141; 61142; 61143;
2136; 26350; 61144; 2177; 56093; 61145; 26374; 26397; 61146; 61147;
61148; 26425; 2226; 2227; 2228; 61149; 2236; 26449; 61150; 61151;
61152; 2252; 2253; 61153; 61154; 61155; 61156; 61157; 2259; 2261;
61158; 56213; 23781; 61159; 61160; 61161; 18267; 61162; 61163;
61164; 61165; 61166; 12323; 591; 597; 2294; 61167; 2302; 2331;
61168; 26673; 61169; 61170; 31647; 31648; 31649; 31650; 31651;
61171.
[0259] The following SEQ ID NOs correspond to the polynucleotides
encoding stomach-specific proteins as described in Table 67A
identified using SBS: 32377; 61246; 61247; 61248; 61249; 61250;
61251; 13311; 13312; 61252; 61253; 61254; 15987; 15988; 27332;
27333; 27334; 61255; 61256; 61257; 61258; 27335; 27336; 61259;
61260; 61261; 61262; 61263; 61264; 61265; 61266; 61267; 61268;
61269; 61270; 61271; 61272; 61273; 61274; 61275; 61276; 27340;
27341; 27342; 27343; 27344; 27345; 27346; 27347; 61277; 61278;
61279; 61280; 61281; 61282; 61283; 61284; 61285; 61286; 61287;
61288; 61289; 61290; 61291; 61292; 61293; 61294; 61295; 5479; 5480;
5481; 5482; 5483; 5485; 5487; 5500; 5510; 5536; 5537; 5539; 5541;
5542; 5543; 5545; 5547; 5550; 5553; 5554; 5555; 5556; 5557; 5558;
5559; 5560; 5561; 5562; 5563; 5564; 5565; 5566; 5567; 5568; 5569;
5570; 5571; 5572; 5573; 61296; 61297; 61298; 5575; 5576; 5577;
5578; 5579; 5580; 5582; 5583; 5591; 5594; 5595; 5596; 5597; 5598;
5599; 5600; 5601; 5602; 5603; 5604; 5605; 5606; 5607; 5608; 5609;
5610; 5611; 5612; 5613; 5614; 5615; 58837; 61299; 27351; 27352;
61300; 61301; 27356; 61302; 27359; 61303.
[0260] The following SEQ ID NOs correspond to the amino acid
sequences of stomach-specific proteins as described in Table 67A
identified using SBS: 32484; 61304; 61305; 61306; 61307; 61308;
61309; 13578; 13579; 61310; 61311; 61312; 16384; 16385; 27362;
27363; 27365; 61313; 61314; 61315; 61316; 27364; 27366; 61317;
61318; 61319; 61320; 61321; 61322; 61323; 61324; 61325; 61326;
61327; 61328; 61329; 61330; 61331; 61332; 61333; 61334; 27370;
27371; 27376; 27377; 27374; 27375; 27372; 27373; 61335; 61336;
61337; 61338; 61339; 61340; 61341; 61342; 61343; 61344; 61345;
61346; 61347; 61348; 61349; 61350; 61351; 61352; 61353; 6313; 6314;
6404; 6316; 6317; 6319; 6388; 6334; 6344; 6370; 6371; 6373; 6375;
6376; 6377; 6379; 6381; 6384; 6387; 6389; 6321; 6390; 6391; 6392;
6393; 6394; 6395; 6397; 6396; 6398; 6399; 6400; 6401; 6402; 6403;
6315; 6405; 6406; 6407; 61354; 61355; 61356; 6409; 6446; 6445;
6444; 6416; 6425; 6433; 6429; 6414; 6417; 6428; 6438; 6431; 6432;
6413; 6434; 6436; 6435; 6412; 6430; 6439; 6440; 6441; 6442; 6443;
6437; 6411; 6410; 6447; 6448; 6449; 58918; 61357; 27381; 27382;
61358; 61359; 27386; 61360; 27389; 61361.
[0261] The following SEQ ID NOs correspond to the polynucleotides
encoding testes-specific proteins as described in Table 68A
identified using SBS: 61484; 61485; 61486; 61487; 61488; 61489;
61490; 27425; 61491; 8122; 8123; 8124; 8125; 8126; 8127; 8128;
8129; 8130; 27426; 61492; 61493; 27427; 61494; 61495; 27428; 27429;
61496; 27430; 61497; 20136; 20137; 5088; 5090; 17502; 61498; 61499;
61500; 61501; 61502; 27435; 27436; 27437; 27438; 27439; 61503;
61504; 25824; 27441; 27442; 53048; 61505; 61506; 27447; 27448;
8167; 61507; 27449; 27450; 61508; 61509; 61510; 61511; 27452; 1813;
27453; 30470; 61512; 21469; 2638; 2639; 61513; 61514; 61515; 27457;
61516; 27458; 27460; 61517; 12678; 2645; 27462; 27463; 12680;
12681; 5118; 61518; 61519; 27468; 27469; 27470; 27472; 27473;
27474; 27475; 27476; 13277; 13278; 27477; 5126; 61520; 8218; 27481;
27482; 61521; 61522; 61523; 18967; 27483; 27484; 24735; 27485;
27486; 27487; 27490; 27489; 61524; 61525; 61526; 61527; 61528;
61529; 61530; 61531; 27492; 27493; 61532; 61533; 61534; 61535;
24739; 61536; 61537; 61538; 27497; 27498; 61539; 61540; 61541;
61542; 27499; 61543; 61544; 61545; 21488; 21489; 27501; 27502;
61546; 61547; 27503; 61548; 61549; 61550; 61551; 27505; 61552;
8243; 61553; 27507; 27508; 61554; 61555; 5131; 27509; 61556; 61557;
27510; 27511; 61558; 27513; 61559; 61560; 61561; 61562; 61563;
61564; 61565; 61566; 61567; 27516; 61568; 61569; 27517; 12688;
61570; 61571; 2662; 27518; 61572; 27519; 61573; 61574; 17531; 8246;
27521; 61575; 27523; 27524; 61576; 61577; 13304; 61578; 27525;
61579; 61580; 61581; 27527; 61582; 27528; 61583; 61584; 61585;
27530; 27531; 27533; 61586; 5135; 61587; 27535; 61588; 61589;
61590; 27537; 27538; 27539; 61591; 27540; 27541; 27542; 27544;
61592; 61593; 61594; 61595; 61596; 61597; 61598; 61599; 61600;
61601; 61602; 61603; 61604; 61605; 27546; 3657; 27548; 27549;
27550; 27551; 27552; 27553; 27554; 61606; 24744; 61607; 24745;
61608; 61609; 27559; 61610; 27560; 61611; 61612; 61613; 61614;
61615; 27561; 20591; 27562; 27563; 27564; 27565; 27566; 27567;
27568; 27569; 27570; 61616; 61617; 27574; 27575; 27576; 61618;
5162; 20160; 61619; 61620; 2677; 27579; 61621; 27581; 27582; 27583;
27584; 27585; 27586; 27587; 27588; 27589; 27590; 61622; 27591;
27592; 61623; 8278; 53095; 27595; 61624; 27597; 20602; 1075; 15986;
27602; 27604; 27605; 3681; 21525; 61625; 27607; 61626; 61627;
27609; 27610; 27611; 61628; 61629; 61630; 61631; 61632; 61633;
61634; 61635; 61636; 61637; 61638; 61639; 61640; 61641; 15991;
14104; 61642; 23323; 23324; 61643; 27617; 61644; 8317; 17579;
17580; 61645; 27618; 61646; 57536; 61647; 61648; 57537; 57538;
57539; 57540; 61649; 61650; 61651; 61652; 61653; 61654; 61655;
57541; 57542; 57543; 57544; 61656; 27620; 61657; 61658; 27622;
27623; 27624; 27625; 61659; 27629; 30499; 61660; 61661; 61662;
61663; 61664; 61665; 61666; 27632; 21546; 27636; 27637; 8353; 8356;
27641; 13333; 19052; 61667; 61668; 61669; 61670; 61671; 61672;
61673; 31377; 31378; 31379; 61674; 27646; 61675; 61676; 61677;
11981; 21556; 27647; 27648; 61678; 61679; 27649; 27650; 27651;
27653; 61680; 21558; 61681; 31381; 61682; 61683; 27655; 27656;
61684; 27658; 3708; 61685; 61686; 61687; 61688; 61689; 61690;
27661; 27663; 59629; 61691; 61692; 61693; 27670; 27671; 61694;
27672; 27673; 27674; 27675; 61695; 27677; 27676; 61696; 8426;
27678; 61697; 61698; 5237; 27681; 27682; 1105; 1106; 61699; 17611;
27684; 61700; 61701; 61702; 61703; 27690; 61704; 61705; 3731; 3732;
3733; 7300; 27693; 27694; 61706; 61707; 61708; 27695; 14146; 1893;
61709; 61710; 61711; 13347; 61712; 61713; 27696; 61714; 61715;
61716; 27698; 61717; 13350; 61718; 61719; 27699; 61720; 61721;
61722; 8500; 61723; 27708; 27709; 61724; 27712; 61725; 27713;
27715; 61726; 27717; 61727; 27718; 27720; 8507; 27722; 27723;
25382; 61728; 61729; 61730; 27727; 61731; 61732; 25383; 27728;
27729; 27731; 61733; 61734; 27733; 27734; 61735; 61736; 61737;
61738; 27735; 21613; 17625; 17624; 17623; 27737; 27738; 27739;
61739; 27740; 27741; 61740; 27742; 61741; 61742; 61743; 61744;
61745; 27745; 27746; 27747; 61746; 27748; 2721; 27751; 27753;
19080; 61747; 27754; 27755; 27756; 61748; 27757; 27758; 25947;
25948; 27759; 61749; 27763; 27764; 8533; 8535; 8534; 13354; 61750;
61751; 61752; 61753; 61754; 61755; 61756; 27768; 61757; 27770;
61758; 61759; 61760; 61761; 61762; 27771; 5263; 21620; 27773;
61763; 27775; 61764; 61765; 61766; 61767; 61768; 61769; 61770;
61771; 61772; 27777; 27778; 61773; 19091; 61774; 61775; 61776;
27781; 27782; 61777; 61778; 27783; 61779; 61780; 27789; 27790;
27791; 61781; 61782; 27793; 61783; 61784; 61785; 61786; 61787;
61788; 27797; 61789; 61790; 27802; 27803; 27804; 5292; 20662;
61791; 61792; 27805; 27806; 61793; 61794; 27807; 2743; 27810;
61795; 27811; 27812; 61796; 61797; 27814; 61798; 12009; 12010;
61799; 27815; 61800; 16051; 32422; 32423; 27818; 27819; 61801;
61802; 61803; 8650; 61804; 61805; 61806; 27821; 61807; 61808;
27822; 27823; 27824; 27825; 27826; 27827; 27828; 27829; 15458;
61809; 61810; 8657; 8658; 27832; 23402; 23403; 8672; 23404; 167;
27836; 27837; 7357; 61811; 61812; 24810; 2779; 2780; 2781; 61813;
14213; 27847; 8693; 8694; 5359; 26000; 21657; 27849; 61814; 27850;
27854; 8715; 8717; 13385; 27855; 27856; 5379; 5380; 5381; 5382;
5383; 5384; 5385; 5386; 5387; 5388; 5389; 61815; 61816; 61817;
61818; 27859; 61819; 55846; 27861; 27862; 27863; 27864; 23411;
61820; 61821; 27867; 27868; 61822; 61823; 61824; 27870; 61825;
2792; 61826; 61827; 27872; 61828; 27874; 27875; 27878; 61829;
27880; 27881; 27883; 27882; 61830; 61831; 27884; 27885; 27886;
27887; 61832; 27888; 61833; 5404; 61834; 27889; 61835; 61836;
61837; 27890; 61838; 61839; 61840; 61841; 61842; 61843; 61844;
61845; 61846; 27896; 27895; 61847; 27897; 27898; 61848; 27899;
61849; 27900; 61850; 2801; 2802; 61851; 61852; 61853; 27901; 27902;
61854; 61855; 27905; 27906; 27903; 27904; 14232; 61856; 61857;
54818; 54819; 27907; 61858; 61859; 61860; 27908; 61861; 61862;
27911; 12031; 61863; 27912; 61864; 61865; 27914; 27913; 61866;
61867; 27915; 27916; 27917; 61868; 61869; 61870; 61871; 61872;
61873; 61874; 61875; 27918; 27919; 27920; 27921; 27922; 61876;
61877; 61878; 61879; 23419; 61880; 61881; 61882; 8752; 61883;
61884; 61885; 61886; 1948; 61887; 61888; 21697; 61889; 27932;
27933; 61890; 61891; 61892; 61893; 61894; 61895; 61896; 61897;
61898; 61899; 26016; 61900; 27938; 27958; 61901; 27942; 27943;
27946; 27947; 27944; 27945; 61902; 27948; 61903; 61904; 61905;
27950; 61906; 27953; 27954; 61907; 27955; 27956; 27941; 61908;
61909; 61910; 61911; 27964; 61912; 61913; 61914; 61915; 27965;
61916; 61917; 61918; 61919; 61920; 61921; 61922; 61923; 61924;
61925; 61926; 61927; 61928; 61929; 61930; 61931; 61932; 61933;
27969; 61934; 61935; 61936; 61937; 61938; 61939; 61940; 61941;
61942; 61943; 61944; 61945; 61946; 61947; 8774; 61948; 21706;
61949; 61950; 61951; 61952; 61953; 61954; 61955; 61956; 61957;
61958; 61959; 61960; 61961; 61962; 61963; 61964; 61965; 61966;
61967; 61968; 28016; 61969; 61970; 27983; 27984; 27985; 27986;
61971; 27987; 61972; 8809; 61973; 61974; 61975; 3877; 61976; 61977;
61978; 61979; 27988; 27989; 61980; 27990; 61981; 12036; 61982;
61983; 61984; 61985; 61986; 61987; 61988; 61989; 61990; 61991;
61992; 61993; 61994; 61995; 61996; 61997; 61998; 61999; 62000;
3880; 62001; 62002; 62003; 62004; 62005; 27994; 62006; 62007;
62008; 62009; 27995; 28020; 62010; 61282; 61283; 62011; 27998;
62012; 62013; 62014; 62015; 14237; 62016; 62017; 62018; 62019;
62020; 62021; 62022; 62023; 62024; 62025; 62026; 28024; 62027;
62028; 62029; 62030; 62031; 62032; 62033; 62034; 62035; 62036;
62037; 62038; 62039; 62040; 62041; 62042; 62043; 62044; 62045;
62046; 62047; 62048; 62049; 62050; 62051; 62052; 62053; 62054;
62055; 62056; 62057; 62058; 62059; 28001; 62060; 62061; 62062;
62063; 62064; 62065; 62066; 62067; 28002; 62068; 62069; 59300;
62070; 62071; 7376; 62072; 62073; 62074; 28004; 62075; 62076;
62077; 62078; 62079; 62080; 62081; 62082; 28005; 28006; 62083;
62084; 62085; 32266; 32267; 62086; 62087; 62088; 8782; 62089;
62090; 62091; 62092; 62093; 62094; 28009; 62095; 62096; 28012;
62097; 62098; 62099; 62100; 62101; 28014; 28015; 62102; 62103;
27982; 62104; 62105; 62106; 28017; 62107; 62108; 62109; 62110;
28018; 62111; 28019; 62112; 62113; 62114; 62115; 27996; 62116;
62117; 62118; 28022; 62119; 62120; 62121; 62122; 62123; 62124;
62125; 62126; 62127; 62128; 8806; 62129; 62130; 62131; 62132;
62133; 62134; 62135; 27999; 62136; 62137; 62138; 62139; 62140;
62141; 62142; 62143; 62144; 7383; 28027; 28028; 62145; 62146;
28029; 28030; 8780; 28031; 28032; 62147; 62148; 62149; 62150;
62151; 62152; 62153; 62154; 28033; 62155; 62156; 62157; 62158;
62159; 62160; 62161; 62162; 62163; 28036; 28037; 28038; 28039;
28040; 28041; 28042; 62164; 62165; 62166; 62167; 62168; 62169;
62170; 62171; 62172; 28043; 28044; 28045; 62173; 62174; 8812;
28046; 28047; 57212; 62175; 62176; 62177; 62178; 62179; 62180;
62181; 62182; 62183; 62184; 62185; 62186; 62187; 62188; 62189;
28048; 28049; 28050; 28051; 28052; 62190; 62191; 62192; 62193;
62194; 62195; 62196; 62197; 62198; 62199; 62200; 62201; 62202;
62203; 62204; 62205; 62206; 62207; 62208; 62209; 62210; 62211;
62212; 62213; 28063; 62214; 62215; 62216; 62217; 62218; 62219;
62220; 62221; 62222; 62223; 62224; 62225; 28068; 62226; 62227;
62228; 62229; 62230; 62231; 28066; 28067; 28065; 62232; 62233;
32268; 32269; 62234; 62235; 1164; 62236; 62237; 62238; 31425;
19154; 8840; 20720; 62239; 32782; 53265; 28075; 26057; 28076;
62240; 62241; 28077; 28078; 62242; 62243; 62244; 62245; 62246;
62247; 62248; 28079; 28080; 28081; 62249; 62250; 62251; 62252;
28082; 28083; 62253; 62254; 23457; 23458; 23459; 62255; 62256;
62257; 30628; 62258; 15482; 28090; 62259; 62260; 32041; 62261;
62262; 62263; 28095; 62264; 62265; 28097; 28098; 28099; 28100;
62266; 62267; 28101; 8887; 62268; 62269; 28103; 62270; 62271;
62272; 62273; 28104; 26068; 28105; 62274; 62275; 62276; 28106;
8894; 62277; 28107; 62278; 62279; 28109; 62280; 62281; 28115;
17117; 14272; 28118; 62282; 62283; 28119; 28120; 62284; 62285;
28121; 8913; 5554; 5555; 217; 218; 219; 13428; 13429; 28122; 62286;
2846; 2847; 20239; 3926; 8922; 8923; 8924; 8925; 62287; 62288;
62289; 8929; 62290; 62291; 62292; 62293; 5635; 5638; 231; 232;
8957; 8958; 28125; 28126; 1991; 1993; 62294; 62295; 62296; 62297;
62298; 28131; 62299; 28132; 28133; 12810; 62300; 3937; 62301;
62302; 28134; 28135; 28136; 62303; 62304; 62305; 62306; 28140; 236;
62307; 20749; 28144; 62308; 28145; 28149; 62309; 28150; 62310;
62311; 62312; 3948; 28154; 28155; 28156; 28157; 28158; 28159;
62313; 1202; 9000; 62314; 28179; 23490; 62315; 62316; 53337; 62317;
9007; 62318; 28183; 62319; 62320; 9010; 9011; 9012; 62321; 250;
251; 28189; 28191; 28192; 62322; 62323; 28193; 16147; 62324; 62325;
62326; 13443; 28198; 28199; 28200; 28201; 28202; 26135; 62327;
53343; 16151; 16152; 16153; 7437; 62328; 62329; 62330; 62331;
28207; 28209; 28210; 28211; 28212; 28213; 62332; 28215; 28216;
28217; 28218; 62333; 62334; 28221; 62335; 62336; 62337; 62338;
62339; 265; 266; 28223; 28224; 28225; 14343; 14344; 14345; 28226;
62340; 62341; 9098; 62342; 21887; 23511; 23512; 23513; 23514;
23515; 14349; 271; 62343; 274; 17877; 17878; 17879; 17880; 17881;
62344; 62345; 9115; 9116; 2894; 62346; 62347; 62348; 62349; 62350;
4008; 28238; 4010; 62351; 28241; 28242; 28243; 62352; 62353; 62354;
284; 28244; 62355; 28246; 62356; 28248; 26161; 26162; 28249; 28250;
28251; 28252; 28253; 28254; 62357; 28256; 62358; 28257; 62359;
62360; 62361; 62362; 62363; 28258; 28259; 62364; 62365; 62366;
28260; 28261; 28262; 62367; 17888; 62368; 62369; 28265; 62370;
28266; 62371; 28268; 62372; 62373; 62374; 1241; 5749; 1243; 9177;
28275; 62375; 24887; 62376; 52875; 17902; 62377; 62378; 62379;
25450; 28281; 62380; 21920; 62381; 62382; 28286; 28287; 21922;
21923; 62383; 62384; 55311; 28290; 28292; 28294; 62385; 28295;
62386; 24898; 21930; 62387; 62388; 62389; 305; 306; 307; 2058;
2059; 28301; 62390; 62391; 62392; 62393; 28309; 62394; 62395; 308;
309; 62396; 28310; 62397; 62398; 62399; 28311; 28312; 62400; 28313;
62401; 62402; 62403; 7486; 62404; 28314; 62405; 62406; 62407;
28315; 28316; 12129; 28317; 28318; 28319; 28320; 62408; 62409;
62410; 2920; 2921; 26202; 62411; 62412; 24900; 62413; 62414; 62415;
62416; 62417; 62418; 62419; 28324; 28325; 14394; 4061; 28327;
28328; 28329; 62420; 62421; 28330; 5790; 1275; 62422; 4072; 4073;
62423; 28333; 62424; 62425; 62426; 28335; 28336; 62427; 62428;
62429; 17923; 28337; 62430; 62431; 62432; 28338; 28339; 62433;
4076; 62434; 62435; 20818; 28340; 28341; 28343; 62436; 28344;
12848; 28345; 62437; 28346; 62438; 28348; 28350; 62439; 9327;
28351; 28352; 62440; 62441; 62442; 62443; 28355; 28356; 28357;
28358; 28359; 28360; 62444; 62445; 62446; 62447; 62448; 62449;
62450; 62451; 62452; 28362; 62453; 12857; 62454; 62455; 62456;
62457; 62458; 17174; 28365; 62459; 28367; 28368; 5820; 62460;
62461; 28369; 62462; 28370; 28371; 62463; 62464; 62465; 62466;
28372; 62467; 345; 28374; 62468; 28375; 28378; 28379; 4100; 28384;
28385; 62469; 62470; 62471; 62472; 62473; 62474; 62475; 62476;
62477; 62478; 5825; 2958; 5826; 2959; 5827; 28386; 28387; 62479;
28389; 24912; 28390; 62480; 4106; 62481; 62482; 28391; 62483;
62484; 62485; 62486; 12152; 62487; 62488; 62489; 62490; 62491;
25473; 62492; 62493; 62494; 62495; 62496; 20835; 62497; 28396;
28398; 361; 62498; 28403; 12871; 62499; 28404; 62500; 62501; 62502;
62503; 28405; 17180; 17181; 26273; 26274; 62504; 62505; 62506;
62507; 62508; 62509; 28418; 62510; 28419; 62511; 28420; 28421;
28422; 28423; 62512.
[0262] The following SEQ ID NOs correspond to the amino acid
sequences of testes-specific proteins as described in Table 68A
identified using SBS: 62513; 62514; 62515; 62516; 62517; 62518;
62519; 28427; 62520; 9450; 9451; 9452; 9453; 9454; 9455; 9456;
9457; 9458; 28428; 62521; 62522; 28429; 62523; 62524; 28430; 28431;
62525; 28432; 62526; 20277; 20278; 5922; 5924; 17971; 62527; 62528;
62529; 62530; 62531; 28437; 28438; 28439; 28440; 28441; 62532;
62533; 26294; 28443; 28444; 53453; 62534; 62535; 28450; 28449;
9495; 62536; 28451; 28452; 62537; 62538; 62539; 62540; 28454; 2112;
28455; 30733; 62541; 22056; 2994; 2995; 62542; 62543; 62544; 28459;
62545; 28460; 28462; 62546; 12886; 3001; 28464; 28465; 12888;
12889; 5952; 62547; 62548; 28470; 28471; 28472; 28475; 28474;
28476; 28477; 28478; 13544; 13545; 28479; 5960; 62549; 9546; 28483;
28484; 62550; 62551; 62552; 19371; 28486; 28485; 24938; 28488;
28487; 28489; 28492; 28491; 62553; 62554; 62555; 62556; 62557;
62558; 62559; 62560; 28494; 28495; 62561; 62562; 62563; 62564;
24942; 62565; 62566; 62567; 28499; 28500; 62568; 62569; 62570;
62571; 28501; 62572; 62573; 62574; 22076; 22075; 28503; 28504;
62575; 62576; 28505; 62577; 62578; 62579; 62580; 28507; 62581;
9571; 62582; 28509; 28510; 62583; 62584; 5965; 28511; 62585; 62586;
28512; 28513; 62587; 28515; 62588; 62589; 62590; 62591; 62592;
62593; 62594; 62595; 62596; 28518; 62597; 62598; 28519; 12896;
62599; 62600; 3018; 28520; 62601; 28521; 62602; 62603; 18000; 9574;
28523; 62604; 28525; 28526; 62605; 62606; 13571; 62607; 28527;
62608; 62609; 62610; 28529; 62611; 28530; 62612; 62613; 62614;
28532; 28533; 28535; 62615; 5969; 62616; 28537; 62617; 62618;
62619; 28539; 28540; 28541; 62620; 28542; 28543; 28544; 28546;
62621; 62622; 62623; 62624; 62625; 62626; 62627; 62628; 62629;
62630; 62631; 62632; 62633; 62634; 28548; 4206; 28550; 28551;
28552; 28553; 28554; 28555; 28556; 62635; 24948; 62636; 24947;
62637; 62638; 28561; 62639; 28562; 62640; 62641; 62642; 62643;
62644; 28563; 20858; 28564; 28565; 28568; 28567; 28566; 28569;
28570; 28571; 28572; 62645; 62646; 28576; 28577; 28578; 62647;
5996; 20301; 62648; 62649; 3033; 28581; 62650; 28583; 28584; 28585;
28586; 28587; 28588; 28589; 28590; 28591; 28592; 62651; 28593;
28594; 62652; 9606; 53500; 28597; 62653; 28599; 20869; 1358; 16383;
28604; 28606; 28607; 4230; 22112; 62654; 28609; 62655; 62656;
28611; 28612; 28613; 62657; 62658; 62659; 62660; 62661; 62662;
62663; 62664; 62665; 62666; 62667; 62668; 62669; 62670; 16388;
14518; 62671; 23654; 23655; 62672; 28619; 62673; 9645; 18048;
18049; 62674; 28620; 62675; 57654; 62676; 62677; 57655; 57656;
57657; 57658; 62678; 62679; 62680; 62681; 62682; 62683; 62684;
57659; 57660; 57661; 57662; 62685; 28622; 62686; 62687; 28624;
28625; 28627; 29262; 62688; 28631; 30762; 62689; 62690; 62691;
62692; 62693; 62694; 62695; 28634; 22133; 28638; 28639; 9681; 9684;
28643; 13600; 19456; 62696; 62697; 62698; 62699; 62700; 62701;
62702; 31539; 31540; 31541; 62703; 28648; 62704; 62705; 62706;
12237; 22143; 28649; 28650; 62707; 62708; 28651; 28652; 28653;
28655; 62709; 22145; 62710; 31543; 62711; 62712; 28657; 28658;
62713; 28660; 4257; 62714; 62715; 62716; 62717; 62718; 62719;
28663; 28665; 59842; 62720; 62721; 62722; 28672; 28673; 62723;
28674; 28675; 28676; 28677; 62724; 28679; 28678; 62725; 9754;
28680; 62726; 62727; 6071; 28683; 28684; 1388; 1389; 62728; 18080;
28686; 62729; 62730; 62731; 62732; 28692; 62733; 62734; 4281; 4280;
4282; 7630; 28695; 28696; 62735; 62736; 62737; 28697; 14560; 2192;
62738; 62739; 62740; 13614; 62741; 62742; 28698; 62743; 62744;
62745; 28700; 62746; 13617; 62747; 62748; 28701; 62749; 62750;
62751; 9828; 62752; 28710; 28711; 62753; 28714; 62754; 28715;
28722; 62755; 28719; 62756; 28720; 28717; 9835; 28724; 28725;
25522; 62757; 62758; 62759; 28729; 62760; 62761; 25523; 28730;
28731; 28733; 62762; 62763; 28735; 28736; 62764; 62765; 62766;
62767; 28737; 22200; 18094; 18092; 18093; 28739; 29047; 29048;
62768; 28743; 28742; 62769; 28744; 62770; 62771; 62772; 62773;
62774; 28747; 28748; 28749; 62775; 28750; 3077; 28753; 28755;
19484; 62776; 28756; 28757; 28758; 62777; 28759; 28760; 26417;
26418; 28761; 62778; 28765; 62779; 9861; 9863; 9862; 13621; 62780;
62781; 62782; 62783; 62784; 62785; 62786; 28770; 62787; 28772;
62788; 62789; 62790; 62791; 62792; 28773; 6097; 22207; 28775;
62793; 28777; 62794; 62795; 62796; 62797; 62798; 62799; 62800;
62801; 62802; 28779; 28780; 62803; 19495; 62804; 62805; 62806;
28783; 28784; 62807; 62808; 28785; 62809; 62810; 28791; 28792;
28793; 62811; 62812; 28795; 62813; 62814; 62815; 62816; 62817;
62818; 28799; 62819; 62820; 28804; 28805; 28806; 6126; 20929;
62821; 62822; 28807; 28808; 62823; 62824; 28809; 3099; 28812;
62825; 28813; 28814; 28815; 62826; 28816; 62827; 12265; 12266;
62828; 28817; 62829; 16448; 32529; 32530; 28820; 28821; 62830;
62831; 62832; 9978; 62833; 62834; 62835; 28823; 62836; 62837;
28824; 28825; 28826; 28830; 28828; 28829; 28827; 28831; 15634;
62838; 62839; 9985; 9986; 28834; 23733; 23734; 10000; 23735; 535;
28838; 28839; 7687; 62840; 62841; 25013; 3135; 3136; 3137; 62842;
14627; 28849; 10021; 10022; 6207; 62843; 22244; 28851; 62844;
28852; 28856; 10043; 10045; 13652; 28857; 28858; 6213; 6214; 6215;
6216; 6221; 6218; 6219; 6220; 6217; 6222; 6223; 62845; 62846;
62847; 62848; 28861; 62849; 56168; 28863; 28864; 28865; 28866;
23742; 62850; 62851; 28869; 28870; 62852; 62853; 62854; 28872;
62855; 3148; 62856; 62857; 28874; 62858; 28876; 28877; 28880;
62859; 28882; 28883; 28885; 28884; 62860; 62861; 28886; 28888;
28887; 28889; 62862; 28890; 62863; 6238; 62864; 28891; 62865;
62866; 62867; 28892; 62868; 62869; 62870; 62871; 62872; 62873;
62874; 62875; 62876; 28898; 28897; 62877; 28900; 28899; 62878;
28901; 62879; 28902; 62880; 3150; 3158; 62881; 62882; 62883; 28904;
28903; 62884; 62885; 28907; 28908; 28905; 28906; 14646; 62886;
62887; 54833; 54834; 28909; 62888; 62889; 62890; 28910; 62891;
62892; 28913; 12287; 62893; 62894; 28914; 62895; 28915; 28916;
62896; 62897; 28918; 28919; 28917; 62898; 62899; 62900; 62901;
62902; 62903; 62904; 62905; 28920; 28921; 28922; 28923; 28924;
62906; 62907; 62908; 62909; 23750; 10080; 62910; 62911; 62912;
62913; 62914; 62915; 62916; 2247; 62917; 62918; 22284; 62919;
28934; 28935; 62920; 62921; 62922; 62923; 62924; 62925; 62926;
62927; 62928; 62929; 26486; 62930; 28940; 28959; 62931; 28944;
62932; 28948; 28946; 62933; 28947; 62934; 28950; 62935; 62936;
62937; 28952; 62938; 28956; 28955; 62939; 28957; 28958; 28961;
62940; 62941; 62942; 62943; 28966; 62944; 62945; 62946; 62947;
28967; 62948; 62949; 62950; 62951; 62952; 62953; 62954; 62955;
62956; 62957; 62958; 62959; 62960; 62961; 62962; 62963; 62964;
62965; 28971; 62966; 62967; 62968; 62969; 62970; 62971; 62972;
62973; 62974; 62975; 62976; 62977; 62978; 62979; 10102; 62980;
22293; 62981; 62982; 62983; 62984; 62985; 62986; 62987; 62988;
62989; 62990; 62991; 62992; 62993; 62994; 62995; 62996; 62997;
62998; 62999; 63000; 28984; 63001; 63002; 28985; 29049; 28987;
28740; 63003; 29019; 63004; 10108; 63005; 63006; 63007; 4429;
63008; 63009; 63010; 63011; 28991; 28990; 63012; 29035; 63013;
12292; 63014; 63015; 63016; 63017; 63018; 63019; 63020; 63021;
63022; 63023; 63024; 63025; 63026; 63027; 63028; 63029; 63030;
63031; 63032; 4426; 63033; 63034; 63035; 63036; 63037; 28996;
63038; 63039; 63040; 63041; 28997; 28998; 63042; 61340; 61341;
63043; 29000; 63044; 63045; 63046; 63047; 14651; 63048; 63049;
63050; 63051; 63052; 63053; 63054; 63055; 63056; 63057; 63058;
29026; 63059; 63060; 63061; 63062; 63063; 63064; 63065; 63066;
63067; 63068; 63069; 63070; 63071; 63072; 63073; 63074; 63075;
63076; 63077; 63078; 63079; 63080; 63081; 63082; 63083; 63084;
63085; 63086; 63087; 63088; 63089; 63090; 63091; 29003; 63092;
63093; 63094; 63095; 63096; 63097; 63098; 63099; 29011; 63100;
63101; 59374; 63102; 63103; 7706; 63104; 63105; 63106; 29006;
63107; 63108; 63109; 63110; 63111; 63112; 63113; 63114; 29007;
29016; 63115; 63116; 63117; 32280; 32281; 63118; 63119; 63120;
10110; 63121; 63122; 63123; 63124; 63125; 28766; 29004; 63126;
63127; 29014; 63128; 63129; 63130; 63131; 63132; 29008; 29017;
63133; 63134; 29018; 63135; 63136; 63137; 28989; 63138; 63139;
63140; 63141; 29020; 63142; 29021; 63143; 63144; 63145; 63146;
29022; 63147; 63148; 63149; 29024; 63150; 63151; 63152; 63153;
63154; 63155; 63156; 63157; 63158; 63159; 10134; 63160; 63161;
63162; 63163; 63164; 63165; 63166; 29001; 63167; 26470; 63168;
63169; 63170; 63171; 63172; 63173; 63174; 7713; 29029; 29030;
63175; 63176; 29031; 29032; 10137; 29033; 29034; 63177; 63178;
63179; 63180; 63181; 63182; 63183; 63184; 28992; 63185; 63186;
63187; 63188; 63189; 63190; 63191; 63192; 63193; 29038; 29039;
29040; 29041; 29042; 29043; 29044; 63194; 63195; 63196; 63197;
63198; 63199; 63200; 63201; 63202; 29045; 29046; 28986; 19552;
19550; 10140; 28741; 28988; 57276; 63203; 63204; 63205; 63206;
63207; 63208; 63209; 63210; 63211; 63212; 63213; 63214; 63215;
63216; 63217; 29050; 29051; 29052; 29053; 29054; 63218; 63219;
63220; 63221; 63222; 63223; 63224; 63225; 63226; 63227; 63228;
63229; 63230; 63231; 63232; 63233; 63234; 63235; 63236; 63237;
63238; 63239; 63240; 63241; 29065; 63242; 63243; 63244; 63245;
63246; 63247; 63248; 63249; 63250; 63251; 63252; 63253; 29070;
63254; 63255; 63256; 63257; 63258; 63259; 29068; 29069; 29067;
63260; 63261; 32282; 32283; 63262; 63263; 1447; 63264; 63265;
63266; 31587; 19558; 10168; 20987; 63267; 32838; 53670; 29077;
26527; 29078; 63268; 63269; 29079; 29080; 63270; 63271; 63272;
63273; 63274; 63275; 63276; 29083; 29082; 29081; 63277; 63278;
63279; 63280; 29084; 29085; 63281; 63282; 23788; 23789; 23790;
63283; 63284; 63285; 30891; 63286; 15658; 29092; 63287; 63288;
32104; 63289; 63290; 63291; 29097; 63292; 63293; 29099; 29100;
29101; 29102; 63294; 63295; 29103; 10215; 63296; 63297; 29105;
63298; 63299; 63300; 63301; 29106; 26538; 29107; 63302; 63303;
63304; 29108; 10222; 63305; 29109; 63306; 63307; 29111; 63308;
63309; 29117; 17277; 14686; 29120; 63310; 63311; 29121; 29122;
63312; 63313; 29123; 10241; 6389; 6321; 586; 587; 585; 13695;
13696; 29124; 63314; 3202; 3203; 20380; 4475; 10250; 10251; 10252;
10253; 63315; 63316; 63317; 10257; 63318; 63319; 63320; 63321;
6469; 6472; 599; 600; 10285; 10286; 29127; 29128; 2290; 2292;
63322; 63323; 63324; 63325; 63326; 29133; 63327; 29134; 29135;
13018; 63328; 4486; 63329; 63330; 29136; 29137; 29138; 63331;
63332; 63333; 63334; 29142; 604; 63335; 21016; 29146; 63336; 29147;
29151; 63337; 29152; 63338; 63339; 63340; 4497; 29156; 29157;
29158; 29159; 29160; 29161; 63341; 1485; 10328; 63342; 29181;
23821; 63343; 63344; 53742; 63345; 10335; 63346; 29185; 63347;
63348; 10338; 10339; 10340; 63349; 618; 619; 29191; 29193; 29194;
63350; 63351; 29195; 16544; 63352; 63353; 63354; 13710; 29200;
29201; 29202; 29203; 29204; 26605; 63355; 53748; 16548; 16549;
16550; 7767; 63356; 63357; 63358; 63359; 29209; 29213; 29214;
29211; 29212; 29215; 63360; 29217; 29218; 29219; 29220; 63361;
63362; 29223; 63363; 63364; 63365; 63366; 63367; 633; 634; 29225;
29226; 29227; 14757; 14758; 14759; 29228; 63368; 63369; 10426;
63370; 22474; 23842; 23843; 23844; 23845; 23846; 14763; 639; 63371;
642; 18346; 18347; 18348; 18349; 18350; 63372; 63373; 10443; 10444;
3250; 63374; 63375; 63376; 63377; 63378; 4557; 29240; 4559; 63379;
29243; 29244; 29245; 63380; 63381; 63382; 652; 29246; 63383; 29248;
63384; 29250; 26631; 26632; 29251; 29254; 29253; 29252; 29255;
29256; 63385; 29258; 63386; 29259; 63387; 63388; 63389; 63390;
63391; 29260; 29261; 63392; 63393; 63394; 28626; 29263; 29264;
63395; 18357; 63396; 63397; 29267; 63398; 29268; 63399; 29270;
63400; 63401; 63402; 1524; 6583; 1526; 10505; 29277; 63403; 25090;
63404; 52890; 18371; 63405; 63406; 63407; 25590; 29283; 63408;
22507; 63409; 63410; 29288; 29289; 22509; 22510; 63411; 63412;
55447; 29292; 29294; 29296; 63413; 29297; 63414; 25101; 22517;
63415; 63416; 63417; 673; 674; 675; 2357; 2358; 29303; 63418;
63419; 63420; 63421; 29311; 63422; 63423; 676; 677; 63424; 29312;
63425; 63426; 63427; 29313; 29314; 63428; 29315; 63429; 63430;
63431; 7816; 63432; 29316; 63433; 63434; 63435; 29317; 29318;
12385; 29319; 29320; 29321; 29322; 63436; 63437; 63438; 3276; 3277;
26672; 63439; 63440; 25103; 63441; 63442; 63443; 63444; 63445;
63446; 63447; 29326; 29327; 14808; 4610; 29329; 29330; 29331;
63448; 63449; 29332; 6624; 1558; 63450; 4621; 4622; 63451; 29335;
63452; 63453; 63454; 29337; 29338; 63455; 63456; 63457; 18392;
29339; 63458; 63459; 63460; 29340; 29341; 63461; 4625; 63462;
63463; 21085; 29342; 29343; 29345; 63464; 29346; 13056; 29347;
63465; 29348; 63466; 29350; 29352; 63467; 10655; 29353; 29354;
63468; 63469; 63470; 63471; 29357; 29358; 29359; 29360; 29361;
29362; 63472; 63473; 63474; 63475; 63476; 63477; 63478; 63479;
63480; 29364; 63481; 13065; 63482; 63483; 63484; 63485; 63486;
17334; 29367; 63487; 29369; 29370; 6654; 63488; 63489; 29371;
63490; 29372; 29373; 63491; 63492; 63493; 63494; 29374; 63495; 713;
29376; 63496; 29377; 29380; 29381; 4649; 29386; 29387; 63497;
63498; 63499; 63500; 63501; 63502; 63503; 63504; 63505; 63506;
6659; 3314; 6661; 3315; 6660; 29388; 29389; 63507; 29391; 25115;
29392; 63508; 4655; 63509; 63510; 29393; 63511; 63512; 63513;
63514; 12408; 63515; 63516; 63517; 63518; 63519; 25613; 63520;
63521; 63522; 63523; 63524; 21102; 63525; 29398; 29400; 729; 63526;
29405; 13079; 63527; 29406; 63528; 63529; 63530; 63531; 29407;
17340; 17341; 26743; 26744; 63532; 63533; 63534; 63535; 63536;
63537; 29420; 63538; 29421; 63539; 29422; 29423; 29424; 29425;
63540.
[0263] The following SEQ ID NOs correspond to the polynucleotides
encoding thymus-specific proteins as described in Table 69A
identified using SBS: 24316; 65194; 65195.
[0264] The following SEQ ID NOs correspond to the amino acid
sequences of thymus-specific proteins as described in Table 69A
identified using SBS: 24446; 65196; 65197.
[0265] The following SEQ ID NOs correspond to the polynucleotides
encoding trachea-specific proteins as described in Table 70A
identified using SBS: 31349; 65203; 65204; 65205; 65206; 65207;
65208; 53071; 3642; 65209; 31849; 65210; 65211; 65212; 65213;
27579; 65214; 65215; 3673; 31854; 65216; 65217; 65218; 65219;
65220; 65221; 11974; 61672; 65222; 17596; 31858; 31859; 65223;
65224; 65225; 19082; 65226; 31861; 1946; 31868; 31869; 31870;
65227; 65228; 65229; 65230; 65231; 65232; 65233; 31871; 31872;
65234; 65235; 65236; 65237; 65238; 65239; 65240; 65241; 65242;
65243; 65244; 65245; 65246; 65247; 31875; 31876; 1970; 65248;
27350; 32043; 20237; 65249; 16114; 16115; 31878; 21792; 21793;
21794; 21795; 21796; 65250; 24402; 65251; 59764; 31879; 31880;
24419; 31882; 65252; 65253; 31884; 65254; 31477; 65255; 65256;
24893; 65257; 9312; 31887; 65258; 65259; 31492; 65260; 65261;
65262; 22004; 22005; 28404.
[0266] The following SEQ ID NOs correspond to the amino acid
sequences of trachea-specific proteins as described in Table 70A
identified using SBS: 31511; 65263; 65264; 65265; 65266; 65267;
65268; 53476; 4191; 65269; 31891; 65270; 65271; 65272; 65273;
28581; 65274; 65275; 4222; 31896; 65276; 65277; 65278; 65279;
65280; 65281; 12230; 62701; 65282; 18065; 31900; 31901; 65283;
65284; 65285; 19486; 65286; 31903; 2245; 31910; 31911; 31912;
65287; 65288; 65289; 65290; 65291; 65292; 65293; 31913; 31914;
65294; 65295; 65296; 65297; 65298; 65299; 65300; 65301; 65302;
65303; 65304; 65305; 65306; 65307; 31917; 31918; 2269; 65308;
27380; 32106; 20378; 65309; 16511; 16512; 31920; 22379; 22380;
22381; 22382; 22383; 65310; 24532; 65311; 59977; 31921; 31922;
24549; 31924; 65312; 65313; 31926; 65314; 31639; 65315; 65316;
25096; 65317; 10640; 31929; 65318; 65319; 31654; 65320; 65321;
65322; 22591; 22592; 29406.
[0267] The following SEQ ID NOs correspond to the polynucleotides
encoding uterus-specific proteins as described in Table 71A
identified using SBS: 27431; 32017; 65466; 32033; 65467; 65468;
65469; 65470; 65471; 65472; 65473; 65474; 65475; 65476; 28302;
28304; 28307; 28308; 65477; 65478; 65479; 32062; 65480.
[0268] The following SEQ ID NOs correspond to the amino acid
sequences of uterus-specific proteins as described in Table 71A
identified using SBS: 28433; 32080; 65481; 32096; 65482; 65483;
65484; 65485; 65486; 65487; 65488; 65489; 65490; 65491; 29304;
29306; 29309; 29310; 65492; 65493; 65494; 32125; 65495.
[0269] The following SEQ ID NOs correspond to the polynucleotides
encoding male organ prostate-specific proteins as described in
Table 72A identified using SBS: 13270; 21443; 3588; 21444; 21445;
17027; 21460; 16; 59267; 3616; 3618; 8196; 15403; 59268; 21492;
21493; 21494; 21495; 21496; 59269; 59270; 53089; 1068; 1847; 1848;
59271; 59272; 59273; 20170; 8343; 59274; 21557; 21558; 21559;
21560; 21561; 20617; 17601; 65511; 65512; 65513; 65514; 65515;
59275; 23359; 59276; 21618; 16037; 59277; 59278; 59279; 21636;
59280; 65516; 20661; 65517; 14189; 65518; 65519; 65520; 65521;
65522; 59281; 3780; 21644; 21645; 59282; 59283; 8668; 59284; 8669;
55235; 55236; 55237; 59285; 21665; 21666; 21667; 21668; 21669;
21670; 21672; 59286; 1159; 1160; 5391; 17103; 5392; 59287; 59288;
59289; 59290; 59291; 59292; 59293; 20702; 59294; 24389; 59295;
59296; 59297; 65523; 65524; 59298; 59299; 65525; 65526; 59300;
21715; 65527; 21717; 59301; 59302; 59303; 21723; 65528; 21724;
21725; 65529; 65530; 65531; 65532; 65533; 65534; 65535; 65536;
65537; 65538; 65539; 65540; 65541; 65542; 65543; 65544; 65545;
65546; 65547; 65548; 65549; 65550; 65551; 65552; 65553; 65554;
65555; 65556; 21726; 21727; 21728; 21729; 21730; 21731; 21732;
21733; 21734; 21735; 21736; 21737; 59304; 59305; 59306; 59307;
21742; 59308; 59309; 59310; 14256; 21786; 21787; 59311; 59312;
59313; 21800; 21801; 21802; 21803; 59314; 59315; 21821; 59316;
21829; 20751; 21838; 59317; 59318; 59319; 59320; 59321; 59322;
21853; 21854; 21855; 21856; 21857; 21858; 21859; 32804; 267; 32325;
32326; 59323; 59324; 59325; 32216; 59326; 59327; 32455; 30675;
59328; 65557; 65558; 21912; 21913; 4028; 59329; 59330; 59331;
59332; 12121; 59333; 21932; 9273; 21939; 57623; 57624; 21960;
59334; 21961; 21972; 59335; 21980; 59336; 32063; 59337; 59338;
59339; 22001; 59340.
[0270] The following SEQ ID NOs correspond to the amino acid
sequences of male organ prostate-specific proteins as described in
Table 72A identified using SBS: 13537; 22030; 4137; 22031; 22032;
17187; 22047; 384; 59341; 4165; 4167; 9524; 15579; 59342; 22079;
22080; 22082; 22081; 22083; 59343; 59344; 53494; 1351; 2147; 2146;
59345; 59346; 59347; 20311; 9671; 59348; 22144; 22145; 22146;
22147; 22148; 20884; 18070; 65559; 65560; 65561; 65562; 65563;
59349; 23690; 59350; 22205; 16434; 59351; 59352; 59353; 22223;
59354; 65564; 20928; 65565; 14604; 65566; 65567; 65568; 65569;
65570; 59355; 4329; 22231; 22232; 59356; 59357; 9996; 59358; 9997;
55371; 55372; 55373; 59359; 22252; 22253; 22254; 22255; 22256;
22257; 22259; 59360; 1442; 1443; 6225; 17263; 6226; 59361; 59362;
59363; 59364; 59365; 59366; 59367; 20969; 59368; 24519; 59369;
59370; 59371; 65571; 65572; 59372; 59373; 65573; 65574; 59374;
22302; 65575; 22304; 59375; 59376; 59377; 22310; 65576; 22311;
22312; 65577; 65578; 65579; 65580; 65581; 65582; 65583; 65584;
65585; 65586; 65587; 65588; 65589; 65590; 65591; 65592; 65593;
65594; 65595; 65596; 65597; 65598; 65599; 65600; 65601; 65602;
65603; 65604; 22313; 22314; 22316; 22315; 22317; 22318; 22319;
22320; 22322; 22321; 22323; 22324; 59378; 59379; 59380; 59381;
22329; 59382; 59383; 59384; 14670; 22373; 22374; 59385; 59386;
59387; 22387; 22388; 22389; 22390; 59388; 59389; 22408; 59390;
22416; 21018; 22425; 59391; 59392; 59393; 59394; 59395; 59396;
22440; 22441; 22442; 22443; 22444; 22445; 22446; 32860; 635; 32343;
32344; 59397; 59398; 59399; 32230; 59400; 59401; 32562; 30938;
59402; 65605; 65606; 22499; 22500; 4577; 59403; 59404; 59405;
59406; 12377; 59407; 22519; 10601; 22526; 57741; 57742; 22547;
59408; 22548; 22559; 59409; 22567; 59410; 32126; 59411; 59412;
59413; 22588; 59414.
[0271] The following SEQ ID NOs correspond to the polynucleotides
encoding male sex organ testes-specific proteins as described in
Table 73A identified using SBS: 61484; 61485; 61486; 61487; 61488;
61489; 61490; 27425; 61491; 8122; 8123; 8124; 8125; 8126; 8127;
8128; 8129; 8130; 27426; 61492; 61493; 27427; 61494; 61495; 27428;
27429; 61496; 27430; 61497; 20136; 20137; 5088; 5090; 17502; 61498;
61499; 61500; 61501; 61502; 27435; 27436; 27437; 65623; 65624;
65625; 27438; 27439; 65626; 61503; 61504; 25824; 27441; 27442;
53048; 61505; 61506; 27447; 27448; 8167; 61507; 27449; 27450;
61508; 61509; 61510; 61511; 27452; 1813; 27453; 30470; 61512;
21469; 2638; 2639; 27455; 61513; 61514; 61515; 27456; 27457; 61516;
27458; 27460; 65627; 61517; 12678; 2645; 27462; 27463; 12680;
12681; 5118; 61518; 61519; 27468; 27469; 27470; 27472; 27473;
27474; 27475; 27476; 13277; 13278; 27477; 5126; 61520; 8218; 27481;
27482; 61521; 61522; 61523; 18967; 27483; 27484; 24735; 27485;
27486; 27487; 27490; 27489; 33; 61524; 61525; 61526; 61527; 61528;
61529; 61530; 61531; 27492; 27493; 61532; 61533; 61534; 61535;
24739; 61536; 61537; 61538; 27497; 27498; 61539; 61540; 61541;
61542; 27499; 61543; 61544; 61545; 21488; 21489; 27501; 27502;
61546; 61547; 27503; 61548; 61549; 61550; 61551; 27505; 61552;
8243; 61553; 27507; 27508; 61554; 61555; 5131; 27509; 61556; 61557;
27510; 27511; 61558; 27513; 61559; 61560; 61561; 61562; 61563;
61564; 61565; 61566; 61567; 27516; 61568; 61569; 27517; 12688;
61570; 61571; 65628; 65629; 2662; 27518; 61572; 27519; 61573;
61574; 17531; 8246; 27521; 61575; 27523; 27524; 61576; 61577;
13304; 61578; 27525; 61579; 61580; 61581; 27527; 61582; 27528;
61583; 61584; 61585; 65630; 65631; 65632; 65633; 27530; 27531;
27533; 61586; 65634; 5135; 61587; 27535; 61588; 61589; 61590;
27537; 27538; 27539; 61591; 27540; 27541; 27542; 27544; 61592;
61593; 61594; 61595; 61596; 61597; 61598; 61599; 61600; 61601;
61602; 61603; 61604; 61605; 27546; 3657; 27548; 27549; 27550;
27551; 27552; 27553; 27554; 61606; 24744; 61607; 24745; 61608;
61609; 27559; 65635; 61610; 27560; 61611; 61612; 61613; 61614;
61615; 27561; 20591; 27562; 27563; 27564; 27565; 27566; 27567;
27568; 27569; 27570; 61616; 61617; 27574; 27575; 27576; 61618;
5162; 20160; 65636; 61619; 61620; 2677; 27579; 61621; 27581; 27582;
27583; 27584; 27585; 27586; 27587; 27588; 27589; 27590; 61622;
27591; 27592; 61623; 8278; 53095; 27595; 61624; 27597; 20602; 1075;
15986; 27602; 27604; 27605; 3681; 21523; 21524; 21525; 61625;
27607; 61626; 61627; 27609; 27610; 27611; 61628; 61629; 61630;
61631; 61632; 61633; 61634; 61635; 61636; 61637; 61638; 61639;
61640; 61641; 15991; 14104; 21535; 61642; 23323; 23324; 61643;
27617; 61644; 8317; 17579; 17580; 61645; 27618; 8323; 61646; 57536;
61647; 61648; 57537; 57538; 57539; 57540; 61649; 61650; 61651;
61652; 61653; 61654; 61655; 57541; 57542; 57543; 57544; 61656;
27620; 61657; 61658; 27622; 27623; 27624; 27625; 61659; 27629;
8336; 30499; 61660; 61661; 61662; 61663; 61664; 61665; 61666;
27632; 21546; 27636; 27637; 8353; 65637; 8356; 27641; 13333; 19052;
61667; 61668; 61669; 61670; 61671; 61672; 61673; 31377; 31378;
31379; 61674; 27646; 61675; 61676; 61677; 11981; 21556; 27647;
27648; 61678; 61679; 27649; 27650; 27651; 27653; 61680; 21558;
61681; 31381; 61682; 61683; 27655; 27656; 61684; 27658; 3708;
61685; 61686; 65638; 61687; 61688; 61689; 61690; 27661; 27663;
59629; 61691; 61692; 61693; 27670; 27671; 61694; 27672; 27673;
27674; 8418; 8419; 27675; 61695; 27677; 27676; 61696; 21578; 8426;
27678; 61697; 61698; 5237; 27681; 27682; 1105; 1106; 1890; 61699;
17611; 27684; 20183; 20184; 61700; 61701; 61702; 61703; 27690;
61704; 61705; 3731; 3732; 3733; 7300; 27692; 27693; 27694; 61706;
61707; 61708; 27695; 14146; 1893; 61709; 61710; 61711; 13347;
61712; 61713; 27696; 61714; 61715; 61716; 27698; 61717; 13350;
61718; 61719; 27699; 61720; 61721; 61722; 8500; 61723; 27708;
27709; 61724; 27712; 61725; 27713; 27715; 61726; 27717; 61727;
27718; 27720; 8507; 27722; 27723; 65639; 65640; 65641; 25382;
61728; 61729; 61730; 27727; 61731; 61732; 25383; 27728; 27729;
57174; 27731; 61733; 61734; 27733; 27734; 61735; 61736; 61737;
61738; 27735; 21613; 17625; 17624; 17623; 27737; 27738; 27739;
61739; 27740; 27741; 61740; 27742; 61741; 61742; 61743; 61744;
61745; 27745; 27746; 27747; 61746; 27748; 2721; 27751; 27753;
19080; 61747; 27754; 27755; 27756; 61748; 27757; 27758; 25947;
25948; 27759; 61749; 27763; 27764; 8533; 8535; 8534; 13354; 61750;
61751; 61752; 61753; 61754; 61755; 61756; 27768; 61757; 27770;
61758; 61759; 61760; 61761; 61762; 27771; 5263; 21620; 27773;
61763; 27775; 61764; 61765; 61766; 61767; 61768; 61769; 61770;
61771; 61772; 27777; 27778; 61773; 19091; 61774; 61775; 61776;
27781; 27782; 61777; 61778; 27783; 61779; 61780; 27789; 27790;
27791; 61781; 61782; 27793; 61783; 61784; 61785; 61786; 61787;
61788; 27797; 61789; 61790; 27802; 27803; 27804; 5292; 20662;
61791; 61792; 27805; 27806; 61793; 61794; 27807; 2743; 27810;
61795; 27811; 27812; 61796; 61797; 27814; 61798; 12009; 12010;
61799; 27815; 61800; 16051; 32422; 32423; 27818; 27819; 61801;
61802; 61803; 27820; 8650; 61804; 61805; 61806; 65642; 27821;
61807; 61808; 27822; 27823; 27824; 27825; 27826; 27827; 27828;
27829; 15458; 61809; 61810; 8657; 8658; 27832; 23402; 23403; 8672;
23404; 167; 27836; 27837; 7357; 61811; 61812; 24810; 2779; 2780;
2781; 61813; 14213; 27847; 8693; 8694; 5359; 26000; 21657; 27849;
61814; 27850; 27854; 8715; 8717; 13385; 27855; 27856; 5379; 5380;
5381; 5382; 5383; 5384; 5385; 5386; 5387; 5388; 5389; 61815; 61816;
61817; 61818; 27859; 61819; 55846; 27860; 27861; 27862; 27863;
27864; 23411; 61820; 61821; 27867; 27868; 61822; 61823; 61824;
27870; 61825; 2792; 61826; 61827; 27872; 61828; 27874; 27875;
27878; 61829; 27880; 27881; 27883; 27882; 61830; 61831; 27884;
27885; 27886; 27887; 61832; 27888; 61833; 5404; 61834; 27889;
61835; 61836; 61837; 27890; 61838; 32265; 61839; 61840; 61841;
61842; 61843; 61844; 61845; 61846; 27896; 27895; 61847; 27897;
27898; 61848; 27899; 61849; 27900; 61850; 2801; 2802; 61851; 61852;
61853; 27901; 27902; 61854; 61855; 27905; 27906; 27903; 27904;
14232; 61856; 61857; 54818; 54819; 27907; 61858; 61859; 61860;
27908; 61861; 61862; 27911; 12031; 61863; 27912; 61864; 61865;
27914; 27913; 61866; 61867; 27915; 27916; 27917; 61868; 61869;
61870; 61871; 61872; 61873; 61874; 61875; 27918; 27919; 27920;
27921; 27922; 27924; 61876; 61877; 61878; 61879; 23419; 61880;
61881; 61882; 8752; 61883; 61884; 61885; 61886; 1948; 61887; 61888;
21697; 61889; 27932; 27933; 61890; 61891; 61892; 61893; 61894;
61895; 61896; 61897; 61898; 61899; 26016; 61900; 27938; 27958;
61901; 27942; 27943; 27946; 27947; 27944; 27945; 61902; 27948;
61903; 61904; 65643; 65644; 61905; 27950; 61906; 27953; 27954;
61907; 27955; 27956; 27941; 61908; 61909; 61910; 61911; 27964;
61912; 61913; 61914; 61915; 27965; 61916; 61917; 61918; 61919;
61920; 61921; 61922; 61923; 61924; 61925; 61926; 61927; 61928;
61929; 61930; 61931; 61932; 61933; 27969; 61934; 61935; 61936;
61937; 61938; 61939; 61940; 61941; 61942; 61943; 61944; 61945;
61946; 61947; 8774; 61948; 21706; 61949; 61950; 61951; 61952;
61953; 61954; 61955; 61956; 61957; 61958; 61959; 61960; 61961;
61962; 61963; 61964; 61965; 61966; 61967; 61968; 28016; 61969;
61970; 27983; 27984; 27985; 27986; 61971; 27987; 61972; 8809;
61973; 61974; 61975; 3877; 61976; 65645; 61977; 61978; 61979;
27988; 27989; 61980; 27990; 61981; 12036; 61982; 61983; 61984;
61985; 61986; 61987; 61988; 61989; 61990; 61991; 61992; 61993;
61994; 61995; 61996; 61997; 61998; 61999; 62000; 3880; 62001;
62002; 62003; 62004; 62005; 27994; 62006; 62007; 62008; 62009;
27995; 28020; 62010; 61282; 61283; 62011; 27998; 62012; 62013;
62014; 62015; 14237; 62016; 62017; 62018; 62019; 62020; 62021;
62022; 62023; 62024; 62025; 62026; 28024; 62027; 62028; 62029;
62030; 62031; 62032; 62033; 62034; 62035; 62036; 62037; 62038;
62039; 62040; 62041; 62042; 62043; 62044; 62045; 62046; 62047;
62048; 62049; 62050; 62051; 62052; 62053; 62054; 62055; 62056;
62057; 62058; 62059; 28001; 62060; 62061; 62062; 62063; 62064;
62065; 62066; 62067; 28002; 65646; 62068; 62069; 59300; 62070;
62071; 7376; 62072; 62073; 62074; 28004; 62075; 62076; 62077;
62078; 62079; 62080; 62081; 62082; 28005; 28006; 62083; 62084;
62085; 32266; 32267; 62086; 62087; 62088; 8782; 62089; 62090;
62091; 62092; 62093; 62094; 28009; 62095; 62096; 28012; 62097;
62098; 62099; 62100; 62101; 28014; 28015; 62102; 62103; 27982;
62104; 62105; 62106; 28017; 62107; 62108; 62109; 62110; 28018;
62111; 28019; 62112; 62113; 65647; 62114; 62115; 27996; 62116;
23444; 62117; 62118; 28022; 62119; 62120; 62121; 62122; 62123;
62124; 62125; 62126; 62127; 62128; 8806; 62129; 62130; 62131;
65648; 62132; 62133; 62134; 62135; 27999; 62136; 62137; 62138;
62139; 62140; 62141; 62142; 62143; 62144; 65649; 7383; 28027;
28028; 62145; 62146; 28029; 28030; 8780; 28031; 28032; 62147;
62148; 62149; 62150; 62151; 62152; 62153; 62154; 28033; 62155;
62156; 62157; 62158; 62159; 62160; 62161; 62162; 62163; 28036;
28037; 28038; 28039; 28040; 28041; 28042; 62164; 62165; 62166;
62167; 62168; 62169; 62170; 62171; 62172; 28043; 28044; 28045;
62173; 62174; 8812; 28046; 28047; 57212; 62175; 62176; 62177;
62178; 62179; 62180; 62181; 62182; 62183; 62184; 62185; 62186;
62187; 62188; 62189; 28048; 28049; 28050; 28051; 28052; 62190;
62191; 62192; 62193; 62194; 62195; 62196; 62197; 62198; 62199;
62200; 62201; 62202; 62203; 62204; 62205; 62206; 62207; 62208;
62209; 62210; 62211; 62212; 62213; 28063; 62214; 62215; 62216;
62217; 62218; 62219; 62220; 62221; 62222; 62223; 62224; 62225;
28068; 62226; 62227; 62228; 62229; 62230; 62231; 28066; 28067;
28065; 62232; 62233; 32268; 32269; 62234; 65650; 62235; 1164;
62236; 62237; 62238; 31425; 19154; 8840; 20720; 62239; 32782;
53265; 28075; 26057; 28076; 62240; 62241; 28077; 28078; 62242;
62243; 62244; 62245; 62246; 62247; 62248; 28079; 28080; 28081;
62249; 62250; 62251; 62252; 28082; 28083; 62253; 62254; 23457;
23458; 23459; 62255; 62256; 62257; 30628; 62258; 65651; 15482;
28090; 62259; 62260; 32041; 62261; 62262; 62263; 28095; 62264;
62265; 28097; 28098; 28099; 28100; 62266; 62267; 28101; 8887;
62268; 62269; 28103; 62270; 62271; 62272; 62273; 28104; 26068;
28105; 62274; 62275; 62276; 65652; 28106; 8894; 62277; 28107;
62278; 62279; 28109; 62280; 62281; 28115; 17117; 14272; 28118;
62282; 62283; 28119; 28120; 62284; 62285; 28121; 8913; 12056;
12057; 12058; 12059; 12060; 5554; 5555; 217; 218; 219; 13428;
13429; 28122; 62286; 2846; 2847; 20239; 3926; 8922; 8923; 8924;
8925; 62287; 62288; 62289; 8929; 62290; 62291; 62292; 62293; 5635;
5638; 231; 232; 8957; 8958; 28125; 28126; 1991; 1993; 62294; 62295;
62296; 62297; 62298; 28131; 62299; 28132; 28133; 12810; 62300;
3937; 62301; 62302; 28134; 28135; 28136; 62303; 62304; 62305;
62306; 28140; 236; 62307; 20749; 28144; 62308; 28145; 28148; 28149;
62309; 28150; 62310; 62311; 62312; 3948; 28154; 28155; 28156;
28157; 28158; 28159; 62313; 65653; 1202; 9000; 62314; 28179; 23490;
62315; 62316; 53337; 62317; 9007; 62318; 28183; 62319; 62320; 9010;
9011; 9012; 62321; 250; 251; 28189; 28191; 28192; 62322; 62323;
28193; 16147; 62324; 62325; 62326; 13443; 28198; 28199; 28200;
28201; 28202; 26135; 62327; 53343; 16151; 16152; 16153; 7437;
62328; 62329; 62330; 62331; 65654; 65655; 28207; 28209; 28210;
28211; 28212; 28213; 62332; 28215; 28216; 28217; 28218; 62333;
62334; 28221; 28222; 55937; 62335; 62336; 7445; 62337; 62338;
62339; 265; 266; 28223; 28224; 28225; 14343; 14344; 14345; 28226;
62340; 62341; 9098; 62342; 21887; 23511; 23512; 23513; 23514;
23515; 14349; 271; 62343; 1228; 274; 17877; 17878; 17879; 17880;
17881; 62344; 62345; 9115; 9116; 2894; 62346; 62347; 28235; 28236;
62348; 62349; 62350; 4008; 28238; 4010; 62351; 28241; 28242; 28243;
62352; 62353; 62354; 284; 28244; 62355; 28246; 62356; 28248; 26161;
26162; 28249; 28250; 28251; 28252; 28253; 28254; 62357; 28256;
62358; 28257; 62359; 65656; 65657; 62360; 62361; 62362; 62363;
28258; 28259; 62364; 62365; 62366; 28260; 28261; 28262; 62367;
17888; 62368; 62369; 28265; 62370; 28266; 62371; 28268; 62372;
62373; 62374; 1241; 5749; 1242; 1243; 9177; 28275; 62375; 24887;
62376; 52875; 17902; 62377; 62378; 62379; 25450; 28281; 62380;
21920; 62381; 62382; 28285; 28286; 28287; 21922; 21923; 62383;
62384; 55311; 28290; 28292; 53396; 28294; 62385; 28295; 62386;
24898; 21930; 62387; 62388; 62389; 305; 306; 307; 2058; 2059;
28301; 62390; 62391; 62392; 62393; 28309; 62394; 62395; 65658;
65659; 308; 309; 62396; 28310; 62397; 62398; 62399; 28311; 28312;
62400; 28313; 62401; 62402; 62403; 7486; 62404; 28314; 62405;
62406; 62407; 28315; 28316; 12129; 28317; 28318; 28319; 28320;
62408; 62409; 62410; 2920; 2921; 26202; 62411; 62412; 24900; 62413;
62414; 62415; 62416; 62417; 62418; 28323; 62419; 28324; 28325;
14394; 4061; 28327; 28328; 28329; 62420; 62421; 28330; 5790; 1275;
62422; 4072; 4073; 62423; 28333; 62424; 62425; 62426; 28335; 28336;
62427; 62428; 62429; 17923; 28337; 62430; 62431; 62432; 28338;
28339; 62433; 4076; 62434; 62435; 20818; 28340; 28341; 28343;
62436; 28344; 12848; 28345; 62437; 28346; 62438; 28348; 28350;
62439; 9327; 28351; 28352; 62440; 62441; 62442; 62443; 28355;
28356; 28357; 28358; 28359; 28360; 62444; 62445; 62446; 62447;
62448; 62449; 62450; 62451; 62452; 28362; 62453; 12857; 62454;
62455; 62456; 62457; 62458; 17174; 28365; 65660; 62459; 28367;
28368; 5820; 62460; 62461; 28369; 62462; 28370; 28371; 62463;
62464; 62465; 62466; 28372; 62467; 345; 28374; 62468; 28375; 28378;
28379; 4100; 28384; 28385; 62469; 65661; 62470; 62471; 62472;
62473; 62474; 62475; 62476; 62477; 62478; 5825; 2958; 5826; 2959;
5827; 28386; 28387; 62479; 28389; 24912; 28390; 62480; 4106; 62481;
62482; 28391; 62483; 62484; 62485; 12151; 62486; 12152; 62487;
62488; 62489; 62490; 62491; 25473; 62492; 62493; 62494; 62495;
62496; 20835; 62497; 28396; 28398; 361; 62498; 4112; 28403; 12871;
62499; 28404; 62500; 62501; 62502; 62503; 28405; 17180; 17181;
26273; 26274; 62504; 62505; 62506; 62507; 62508; 65662; 62509;
22021; 28418; 62510; 28419; 62511; 28420; 28421; 28422; 28423;
62512.
[0272] The following SEQ ID NOs correspond to the amino acid
sequences of male sex organ testes-specific proteins as described
in Table 73A identified using SBS: 62513; 62514; 62515; 62516;
62517; 62518; 62519; 28427; 62520; 9450; 9451; 9452; 9453; 9454;
9455; 9456; 9457; 9458; 28428; 62521; 62522; 28429; 62523; 62524;
28430; 28431; 62525; 28432; 62526; 20277; 20278; 5922; 5924; 17971;
62527; 62528; 62529; 62530; 62531; 28437; 28438; 28439; 65663;
65664; 65665; 28440; 28441; 65666; 62532; 62533; 26294; 28443;
28444; 53453; 62534; 62535; 28450; 28449; 9495; 62536; 28451;
28452; 62537; 62538; 62539; 62540; 28454; 2112; 28455; 30733;
62541; 22056; 2994; 2995; 28457; 62542; 62543; 62544; 28458; 28459;
62545; 28460; 28462; 65667; 62546; 12886; 3001; 28464; 28465;
12888; 12889; 5952; 62547; 62548; 28470; 28471; 28472; 28475;
28474; 28476; 28477; 28478; 13544; 13545; 28479; 5960; 62549; 9546;
28483; 28484; 62550; 62551; 62552; 19371; 28486; 28485; 24938;
28488; 28487; 28489; 28492; 28491; 401; 62553; 62554; 62555; 62556;
62557; 62558; 62559; 62560; 28494; 28495; 62561; 62562; 62563;
62564; 24942; 62565; 62566; 62567; 28499; 28500; 62568; 62569;
62570; 62571; 28501; 62572; 62573; 62574; 22076; 22075; 28503;
28504; 62575; 62576; 28505; 62577; 62578; 62579; 62580; 28507;
62581; 9571; 62582; 28509; 28510; 62583; 62584; 5965; 28511; 62585;
62586; 28512; 28513; 62587; 28515; 62588; 62589; 62590; 62591;
62592; 62593; 62594; 62595; 62596; 28518; 62597; 62598; 28519;
12896; 62599; 62600; 65668; 65669; 3018; 28520; 62601; 28521;
62602; 62603; 18000; 9574; 28523; 62604; 28525; 28526; 62605;
62606; 13571; 62607; 28527; 62608; 62609; 62610; 28529; 62611;
28530; 62612; 62613; 62614; 65670; 65671; 65672; 65673; 28532;
28533; 28535; 62615; 65674; 5969; 62616; 28537; 62617; 62618;
62619; 28539; 28540; 28541; 62620; 28542; 28543; 28544; 28546;
62621; 62622; 62623; 62624; 62625; 62626; 62627; 62628; 62629;
62630; 62631; 62632; 62633; 62634; 28548; 4206; 28550; 28551;
28552; 28553; 28554; 28555; 28556; 62635; 24948; 62636; 24947;
62637; 62638; 28561; 65675; 62639; 28562; 62640; 62641; 62642;
62643; 62644; 28563; 20858; 28564; 28565; 28568; 28567; 28566;
28569; 28570; 28571; 28572; 62645; 62646; 28576; 28577; 28578;
62647; 5996; 20301; 65676; 62648; 62649; 3033; 28581; 62650; 28583;
28584; 28585; 28586; 28587; 28588; 28589; 28590; 28591; 28592;
62651; 28593; 28594; 62652; 9606; 53500; 28597; 62653; 28599;
20869; 1358; 16383; 28604; 28606; 28607; 4230; 22110; 22111; 22112;
62654; 28609; 62655; 62656; 28611; 28612; 28613; 62657; 62658;
62659; 62660; 62661; 62662; 62663; 62664; 62665; 62666; 62667;
62668; 62669; 62670; 16388; 14518; 22122; 62671; 23654; 23655;
62672; 28619; 62673; 9645; 18048; 18049; 62674; 28620; 9651; 62675;
57654; 62676; 62677; 57655; 57656; 57657; 57658; 62678; 62679;
62680; 62681; 62682; 62683; 62684; 57659; 57660; 57661; 57662;
62685; 28622; 62686; 62687; 28624; 28625; 28627; 29262; 62688;
28631; 9664; 30762; 62689; 62690; 62691; 62692; 62693; 62694;
62695; 28634; 22133; 28638; 28639; 9681; 65677; 9684; 28643; 13600;
19456; 62696; 62697; 62698; 62699; 62700; 62701; 62702; 31539;
31540; 31541; 62703; 28648; 62704; 62705; 62706; 12237; 22143;
28649; 28650; 62707; 62708; 28651; 28652; 28653; 28655; 62709;
22145; 62710; 31543; 62711; 62712; 28657; 28658; 62713; 28660;
4257; 62714; 62715; 65678; 62716; 62717; 62718; 62719; 28663;
28665; 59842; 62720; 62721; 62722; 28672; 28673; 62723; 28674;
28675; 28676; 9746; 9747; 28677; 62724; 28679; 28678; 62725; 22165;
9754; 28680; 62726; 62727; 6071; 28683; 28684; 1388; 1389; 2189;
62728; 18080; 28686; 20324; 20325; 62729; 62730; 62731; 62732;
28692; 62733; 62734; 4281; 4280; 4282; 7630; 28694; 28695; 28696;
62735; 62736; 62737; 28697; 14560; 2192; 62738; 62739; 62740;
13614; 62741; 62742; 28698; 62743; 62744; 62745; 28700; 62746;
13617; 62747; 62748; 28701; 62749; 62750; 62751; 9828; 62752;
28710; 28711; 62753; 28714; 62754; 28715; 28722; 62755; 28719;
62756; 28720; 28717; 9835; 28724; 28725; 65679; 65680; 65681;
25522; 62757; 62758; 62759; 28729; 62760; 62761; 25523; 28730;
28731; 57238; 28733; 62762; 62763; 28735; 28736; 62764; 62765;
62766; 62767; 28737; 22200; 18094; 18092; 18093; 28739; 29047;
29048; 62768; 28743; 28742; 62769; 28744; 62770; 62771; 62772;
62773; 62774; 28747; 28748; 28749; 62775; 28750; 3077; 28753;
28755; 19484; 62776; 28756; 28757; 28758; 62777; 28759; 28760;
26417; 26418; 28761; 62778; 28765; 62779; 9861; 9863; 9862; 13621;
62780; 62781; 62782; 62783; 62784; 62785; 62786; 28770; 62787;
28772; 62788; 62789; 62790; 62791; 62792; 28773; 6097; 22207;
28775; 62793; 28777; 62794; 62795; 62796; 62797; 62798; 62799;
62800; 62801; 62802; 28779; 28780; 62803; 19495; 62804; 62805;
62806; 28783; 28784; 62807; 62808; 28785; 62809; 62810; 28791;
28792; 28793; 62811; 62812; 28795; 62813; 62814; 62815; 62816;
62817; 62818; 28799; 62819; 62820; 28804; 28805; 28806; 6126;
20929; 62821; 62822; 28807; 28808; 62823; 62824; 28809; 3099;
28812; 62825; 28813; 28814; 28815; 62826; 28816; 62827; 12265;
12266; 62828; 28817; 62829; 16448; 32529; 32530; 28820; 28821;
62830; 62831; 62832; 28822; 9978; 62833; 62834; 62835; 65682;
28823; 62836; 62837; 28824; 28825; 28826; 28830; 28828; 28829;
28827; 28831; 15634; 62838; 62839; 9985; 9986; 28834; 23733; 23734;
10000; 23735; 535; 28838; 28839; 7687; 62840; 62841; 25013; 3135;
3136; 3137; 62842; 14627; 28849; 10021; 10022; 6207; 62843; 22244;
28851; 62844; 28852; 28856; 10043; 10045; 13652; 28857; 28858;
6213; 6214; 6215; 6216; 6221; 6218; 6219; 6220; 6217; 6222; 6223;
62845; 62846; 62847; 62848; 28861; 62849; 56168; 28862; 28863;
28864; 28865; 28866; 23742; 62850; 62851; 28869; 28870; 62852;
62853; 62854; 28872; 62855; 3148; 62856; 62857; 28874; 62858;
28876; 28877; 28880; 62859; 28882; 28883; 28885; 28884; 62860;
62861; 28886; 28888; 28887; 28889; 62862; 28890; 62863; 6238;
62864; 28891; 62865; 62866; 62867; 28892; 62868; 32279; 62869;
62870; 62871; 62872; 62873; 62874; 62875; 62876; 28898; 28897;
62877; 28900; 28899; 62878; 28901; 62879; 28902; 62880; 3150; 3158;
62881; 62882; 62883; 28904; 28903; 62884; 62885; 28907; 28908;
28905; 28906; 14646; 62886; 62887; 54833; 54834; 28909; 62888;
62889; 62890; 28910; 62891; 62892; 28913; 12287; 62893; 62894;
28914; 62895; 28915; 28916; 62896; 62897; 28918; 28919; 28917;
62898; 62899; 62900; 62901; 62902; 62903; 62904; 62905; 28920;
28921; 28922; 28923; 28924; 28926; 62906; 62907; 62908; 62909;
23750; 10080; 62910; 62911; 62912; 62913; 62914; 62915; 62916;
2247; 62917; 62918; 22284; 62919; 28934; 28935; 62920; 62921;
62922; 62923; 62924; 62925; 62926; 62927; 62928; 62929; 26486;
62930; 28940; 28959; 62931; 28944; 62932; 28948; 28946; 62933;
28947; 62934; 28950; 62935; 62936; 65683; 65684; 62937; 28952;
62938; 28956; 28955; 62939; 28957; 28958; 28961; 62940; 62941;
62942; 62943; 28966; 62944; 62945; 62946; 62947; 28967; 62948;
62949; 62950; 62951; 62952; 62953; 62954; 62955; 62956; 62957;
62958; 62959; 62960; 62961; 62962; 62963; 62964; 62965; 28971;
62966; 62967; 62968; 62969; 62970; 62971; 62972; 62973; 62974;
62975; 62976; 62977; 62978; 62979; 10102; 62980; 22293; 62981;
62982; 62983; 62984; 62985; 62986; 62987; 62988; 62989; 62990;
62991; 62992; 62993; 62994; 62995; 62996; 62997; 62998; 62999;
63000; 28984; 63001; 63002; 28985; 29049; 28987; 28740; 63003;
29019; 63004; 10108; 63005; 63006; 63007; 4429; 63008; 65685;
63009; 63010; 63011; 28991; 28990; 63012; 29035; 63013; 12292;
63014; 63015; 63016; 63017; 63018; 63019; 63020; 63021; 63022;
63023; 63024; 63025; 63026; 63027; 63028; 63029; 63030; 63031;
63032; 4426; 63033; 63034; 63035; 63036; 63037; 28996; 63038;
63039; 63040; 63041; 28997; 28998; 63042; 61340; 61341; 63043;
29000; 63044; 63045; 63046; 63047; 14651; 63048; 63049; 63050;
63051; 63052; 63053; 63054; 63055; 63056; 63057; 63058; 29026;
63059; 63060; 63061; 63062; 63063; 63064; 63065; 63066; 63067;
63068; 63069; 63070; 63071; 63072; 63073; 63074; 63075; 63076;
63077; 63078; 63079; 63080; 63081; 63082; 63083; 63084; 63085;
63086; 63087; 63088; 63089; 63090; 63091; 29003; 63092; 63093;
63094; 63095; 63096; 63097; 63098; 63099; 29011; 65686; 63100;
63101; 59374; 63102; 63103; 7706; 63104; 63105; 63106; 29006;
63107; 63108; 63109; 63110; 63111; 63112; 63113; 63114; 29007;
29016; 63115; 63116; 63117; 32280; 32281; 63118; 63119; 63120;
10110; 63121; 63122; 63123; 63124; 63125; 28766; 29004; 63126;
63127; 29014; 63128; 63129; 63130; 63131; 63132; 29008; 29017;
63133; 63134; 29018; 63135; 63136; 63137; 28989; 63138; 63139;
63140; 63141; 29020; 63142; 29021; 63143; 63144; 65687; 63145;
63146; 29022; 63147; 23775; 63148; 63149; 29024; 63150; 63151;
63152; 63153; 63154; 63155; 63156; 63157; 63158; 63159; 10134;
63160; 63161; 63162; 65688; 63163; 63164; 63165; 63166; 29001;
63167; 26470; 63168; 63169; 63170; 63171; 63172; 63173; 63174;
65689; 7713; 29029; 29030; 63175; 63176; 29031; 29032; 10137;
29033; 29034; 63177; 63178; 63179; 63180; 63181; 63182; 63183;
63184; 28992; 63185; 63186; 63187; 63188; 63189; 63190; 63191;
63192; 63193; 29038; 29039; 29040; 29041; 29042; 29043; 29044;
63194; 63195; 63196; 63197; 63198; 63199; 63200; 63201; 63202;
29045; 29046; 28986; 19552; 19550; 10140; 28741; 28988; 57276;
63203; 63204; 63205; 63206; 63207; 63208; 63209; 63210; 63211;
63212; 63213; 63214; 63215; 63216; 63217; 29050; 29051; 29052;
29053; 29054; 63218; 63219; 63220; 63221; 63222; 63223; 63224;
63225; 63226; 63227; 63228; 63229; 63230; 63231; 63232; 63233;
63234; 63235; 63236; 63237; 63238; 63239; 63240; 63241; 29065;
63242; 63243; 63244; 63245; 63246; 63247; 63248; 63249; 63250;
63251; 63252; 63253; 29070; 63254; 63255; 63256; 63257; 63258;
63259; 29068; 29069; 29067; 63260; 63261; 32282; 32283; 63262;
65690; 63263; 1447; 63264; 63265; 63266; 31587; 19558; 10168;
20987; 63267; 32838; 53670; 29077; 26527; 29078; 63268; 63269;
29079; 29080; 63270; 63271; 63272; 63273; 63274; 63275; 63276;
29083; 29082; 29081; 63277; 63278; 63279; 63280; 29084; 29085;
63281; 63282; 23788; 23789; 23790; 63283; 63284; 63285; 30891;
63286; 65691; 15658; 29092; 63287; 63288; 32104; 63289; 63290;
63291; 29097; 63292; 63293; 29099; 29100; 29101; 29102; 63294;
63295; 29103; 10215; 63296; 63297; 29105; 63298; 63299; 63300;
63301; 29106; 26538; 29107; 63302; 63303; 63304; 65692; 29108;
10222; 63305; 29109; 63306; 63307; 29111; 63308; 63309; 29117;
17277; 14686; 29120; 63310; 63311; 29121; 29122; 63312; 63313;
29123; 10241; 12312; 12313; 12314; 12315; 12316; 6389; 6321; 586;
587; 585; 13695; 13696; 29124; 63314; 3202; 3203; 20380; 4475;
10250; 10251; 10252; 10253; 63315; 63316; 63317; 10257; 63318;
63319; 63320; 63321; 6469; 6472; 599; 600; 10285; 10286; 29127;
29128; 2290; 2292; 63322; 63323; 63324; 63325; 63326; 29133; 63327;
29134; 29135; 13018; 63328; 4486; 63329; 63330; 29136; 29137;
29138; 63331; 63332; 63333; 63334; 29142; 604; 63335; 21016; 29146;
63336; 29147; 29150; 29151; 63337; 29152; 63338; 63339; 63340;
4497; 29156; 29157; 29158; 29159; 29160; 29161; 63341; 65693; 1485;
10328; 63342; 29181; 23821; 63343; 63344; 53742; 63345; 10335;
63346; 29185; 63347; 63348; 10338; 10339; 10340; 63349; 618; 619;
29191; 29193; 29194; 63350; 63351; 29195; 16544; 63352; 63353;
63354; 13710; 29200; 29201; 29202; 29203; 29204; 26605; 63355;
53748; 16548; 16549; 16550; 7767; 63356; 63357; 63358; 63359;
65694; 65695; 29209; 29213; 29214; 29211; 29212; 29215; 63360;
29217; 29218; 29219; 29220; 63361; 63362; 29223; 29224; 56259;
63363; 63364; 7775; 63365; 63366; 63367; 633; 634; 29225; 29226;
29227; 14757; 14758; 14759; 29228; 63368; 63369; 10426; 63370;
22474; 23842; 23843; 23844; 23845; 23846; 14763; 639; 63371; 1511;
642; 18346; 18347; 18348; 18349; 18350; 63372; 63373; 10443; 10444;
3250; 63374; 63375; 29237; 29238; 63376; 63377; 63378; 4557; 29240;
4559; 63379; 29243; 29244; 29245; 63380; 63381; 63382; 652; 29246;
63383; 29248; 63384; 29250; 26631; 26632; 29251; 29254; 29253;
29252; 29255; 29256; 63385; 29258; 63386; 29259; 63387; 65696;
65697; 63388; 63389; 63390; 63391; 29260; 29261; 63392; 63393;
63394; 28626; 29263; 29264; 63395; 18357; 63396; 63397; 29267;
63398; 29268; 63399; 29270; 63400; 63401; 63402; 1524; 6583; 1525;
1526; 10505; 29277; 63403; 25090; 63404; 52890; 18371; 63405;
63406; 63407; 25590; 29283; 63408; 22507; 63409; 63410; 29287;
29288; 29289; 22509; 22510; 63411; 63412; 55447; 29292; 29294;
53801; 29296; 63413; 29297; 63414; 25101; 22517; 63415; 63416;
63417; 673; 674; 675; 2357; 2358; 29303; 63418; 63419; 63420;
63421; 29311; 63422; 63423; 65698; 65699; 676; 677; 63424; 29312;
63425; 63426; 63427; 29313; 29314; 63428; 29315; 63429; 63430;
63431; 7816; 63432; 29316; 63433; 63434; 63435; 29317; 29318;
12385; 29319; 29320; 29321; 29322; 63436; 63437; 63438; 3276; 3277;
26672; 63439; 63440; 25103; 63441; 63442; 63443; 63444; 63445;
63446; 29325; 63447; 29326; 29327; 14808; 4610; 29329; 29330;
29331; 63448; 63449; 29332; 6624; 1558; 63450; 4621; 4622; 63451;
29335; 63452; 63453; 63454; 29337; 29338; 63455; 63456; 63457;
18392; 29339; 63458; 63459; 63460; 29340; 29341; 63461; 4625;
63462; 63463; 21085; 29342; 29343; 29345; 63464; 29346; 13056;
29347; 63465; 29348; 63466; 29350; 29352; 63467; 10655; 29353;
29354; 63468; 63469; 63470; 63471; 29357; 29358; 29359; 29360;
29361; 29362; 63472; 63473; 63474; 63475; 63476; 63477; 63478;
63479; 63480; 29364; 63481; 13065; 63482; 63483; 63484; 63485;
63486; 17334; 29367; 65700; 63487; 29369; 29370; 6654; 63488;
63489; 29371; 63490; 29372; 29373; 63491; 63492; 63493; 63494;
29374; 63495; 713; 29376; 63496; 29377; 29380; 29381; 4649; 29386;
29387; 63497; 65701; 63498; 63499; 63500; 63501; 63502; 63503;
63504; 63505; 63506; 6659; 3314; 6661; 3315; 6660; 29388; 29389;
63507; 29391; 25115; 29392; 63508; 4655; 63509; 63510; 29393;
63511; 63512; 63513; 12407; 63514; 12408; 63515; 63516; 63517;
63518; 63519; 25613; 63520; 63521; 63522; 63523; 63524; 21102;
63525; 29398; 29400; 729; 63526; 4661; 29405; 13079; 63527; 29406;
63528; 63529; 63530; 63531; 29407; 17340; 17341; 26743; 26744;
63532; 63533; 63534; 63535; 63536; 65702; 63537; 22608; 29420;
63538; 29421; 63539; 29422; 29423; 29424; 29425; 63540.
[0273] The following SEQ ID NOs correspond to the polynucleotides
encoding female sex organ, breast-specific proteins as described in
Table 74A identified using SBS: 17025; 65771; 14099; 8321; 54808;
54809; 17063; 17064; 17080; 54810; 54811; 54812; 54813; 54814;
54815; 54816; 54817; 54818; 54819; 32266; 32267; 54820; 54821;
9008; 17132; 65654; 65655; 54822; 17152; 17153; 24429; 17171;
17172.
[0274] The following SEQ ID NOs correspond to the amino acid
sequences of female sex organ, breast-specific proteins as
described in Table 74A identified using SBS: 17185; 65772; 14513;
9649; 54823; 54824; 17223; 17224; 17240; 54825; 54826; 54827;
54828; 54829; 54830; 54831; 54832; 54833; 54834; 32280; 32281;
54835; 54836; 10336; 17292; 65694; 65695; 54837; 17312; 17313;
24559; 17331; 17332.
[0275] The following SEQ ID NOs correspond to the polynucleotides
encoding female sex organ, cervix-specific proteins as described in
Table 75A identified using SBS: 14134; 32022; 54868; 21644; 65777;
65778; 65779; 65780; 65781; 65782; 14256; 54869; 54870; 54871;
65783; 54872; 54873; 54874.
[0276] The following SEQ ID NOs correspond to the amino acid
sequences of female sex organ, cervix-specific proteins as
described in Table 75A identified using SBS: 14548; 32085; 54875;
22231; 65784; 65785; 65786; 65787; 65788; 65789; 14670; 54876;
54877; 54878; 65790; 54879; 54880; 54881.
[0277] The following SEQ ID NOs correspond to the polynucleotides
encoding female sex organ, ovary-specific proteins as described in
Table 76A identified using SBS: 65793; 58733; 58734; 58735; 58736;
14166; 58737; 65642; 27821; 58738; 58739; 65646; 58740; 58741;
65649; 58742; 32270; 28222; 58743; 58744; 58745; 58746.
[0278] The following SEQ ID NOs correspond to the amino acid
sequences of female sex organ, ovary-specific proteins as described
in Table 76A identified using SBS: 65794; 58747; 58748; 58749;
58750; 14580; 58751; 65682; 28823; 58752; 58753; 65686; 58754;
58755; 65689; 58756; 32284; 29224; 58757; 58758; 58759; 58760.
[0279] The following SEQ ID NOs correspond to the polynucleotides
encoding female sex organ, uterus-specific proteins as described in
Table 77A identified using SBS: 27431; 65630; 65631; 65632; 65633;
32017; 65797; 65466; 32033; 65467; 65468; 65469; 65470; 65471;
65472; 65473; 65474; 65475; 65476; 28302; 28304; 28307; 28308;
65477; 65478; 65479; 32062; 65480; 65798.
[0280] The following SEQ ID NOs correspond to the amino acid
sequences of female sex organ, uterus-specific proteins as
described in Table 77A identified using SBS: 28433; 65670; 65671;
65672; 65673; 32080; 65799; 65481; 32096; 65482; 65483; 65484;
65485; 65486; 65487; 65488; 65489; 65490; 65491; 29304; 29306;
29309; 29310; 65492; 65493; 65494; 32125; 65495; 65800.
[0281] The following SEQ ID NOs correspond to the amino acid
sequences of adrenal gland-specific proteins identified using SBS
that have also been identified by mass spectrometry as described in
Table 78A: 52880; 388; 449; 459; 479; 480; 541; 52888; 574; 649;
648; 52890; 52891.
[0282] The following SEQ ID NOs correspond to the amino acid
sequences of bladder-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 53001; 22128; 1470; 1471; 4654.
[0283] The following SEQ ID NOs correspond to the amino acid
sequences of brain-specific proteins identified using SBS that have
also been identified by mass spectrometry as described in Table
78A: 2095; 2096; 2097; 53446; 53449; 53456; 12175; 12176; 53461;
23617; 16313; 53464; 9519; 5946; 3003; 9529; 53469; 3007; 53470;
53481; 3023; 53488; 4208; 5987; 5988; 5989; 53491; 4211; 4213;
4215; 53493; 22102; 53499; 7605; 32081; 53508; 9640; 25504; 53511;
53512; 53513; 9662; 53514; 9665; 53518; 9682; 53520; 20319; 53523;
53524; 53525; 53526; 6057; 6060; 6061; 6064; 53533; 1390; 9809;
53542; 9864; 9868; 9877; 53560; 4304; 53561; 53562; 7662; 7668;
3093; 9908; 9909; 9910; 9911; 53571; 9914; 53574; 53575; 9915;
9916; 53580; 53582; 4336; 53583; 53584; 28820; 28821; 53586; 53590;
9999; 19523; 10019; 10020; 53604; 53605; 53608; 53611; 3141; 53665;
53666; 53667; 29074; 53674; 53675; 13004; 13005; 53676; 53677;
53678; 53679; 53680; 53686; 53687; 53688; 53692; 53693; 53695;
10213; 53700; 10240; 4466; 12317; 53708; 53710; 53712; 1482; 53718;
53719; 13014; 13015; 6470; 25566; 12324; 10277; 53722; 53723;
10278; 4487; 23814; 23815; 53730; 53731; 53733; 17288; 612; 613;
20386; 4503; 53744; 3234; 23837; 53773; 12363; 53777; 53780; 53790;
53791; 29286; 668; 53796; 53798; 13047; 53801; 53802; 3270; 29302;
53808; 25594; 53810; 4608; 4609; 53815; 53816; 3283; 53828; 19710;
3297; 4630; 3307; 53835; 1577; 4641; 53838; 22575; 53839; 3314;
3315.
[0284] The following SEQ ID NOs correspond to the amino acid
sequences of breast-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 17185; 54829; 54831; 54832; 17292; 17312; 17313;
17332.
[0285] The following SEQ ID NOs correspond to the amino acid
sequences of cervix-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 14670; 54876; 54877; 54878; 54880.
[0286] The following SEQ ID NOs correspond to the amino acid
sequences of heart-specific proteins identified using SBS that have
also been identified by mass spectrometry as described in Table
78A: 1313; 14454; 9460; 54932; 4211; 4213; 4215; 14512; 14517;
28771; 14668; 14691; 14702; 14707; 14709; 14710; 54946; 54947;
54948; 54949; 14777; 14787; 20404; 1568; 1569; 1570; 1571; 1572;
1574; 3308; 3309; 3311; 3312.
[0287] The following SEQ ID NOs correspond to the amino acid
sequences of kidney-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 12879; 12880; 55325; 55330; 26339; 23641; 14508; 55340;
55342; 55343; 55345; 55346; 55347; 55351; 55356; 55358; 55362;
55363; 2242; 55364; 55366; 55367; 55368; 55369; 55370; 55371;
55372; 55373; 32834; 32835; 28859; 17261; 17259; 17258; 25018;
20962; 55379; 55380; 55381; 55423; 32543; 55429; 15674; 15675;
15676; 15677; 15678; 31612; 31613; 31614; 31615; 31616; 23817;
15683; 16580; 16581; 55440; 55441; 55448; 3292; 55452; 21083; 2372;
55457; 55458.
[0288] The following SEQ ID NOs correspond to the amino acid
sequences of liver-specific proteins identified using SBS that have
also been identified by mass spectrometry as described in Table
78A: 55998; 56000; 56001; 56002; 56003; 56004; 12162; 56006; 56011;
5922; 1319; 56015; 56016; 56017; 56018; 20280; 17976; 17977; 56019;
9485; 56020; 56022; 15569; 20852; 56023; 56024; 56025; 4147; 386;
56026; 9500; 56027; 56028; 56029; 2113; 56031; 56032; 56033; 56034;
56036; 56037; 56038; 56039; 5976; 56042; 56043; 56044; 56045;
56046; 56047; 56048; 56049; 56050; 56051; 56052; 56053; 31530;
31529; 56059; 19418; 56060; 423; 56061; 56062; 56063; 23646; 23647;
56066; 56067; 56068; 56069; 56070; 56071; 56072; 56074; 56078;
56079; 56087; 24978; 24979; 24980; 2177; 56093; 56096; 20887; 2185;
56099; 13611; 56100; 56102; 56103; 25520; 23688; 56104; 13612;
56106; 56108; 56109; 56110; 56111; 56113; 56114; 56122; 56123;
56128; 56136; 56141; 19509; 56142; 56146; 14612; 56150; 56151;
15629; 56152; 56162; 56163; 56164; 56165; 56167; 15645; 56169;
56209; 56210; 56211; 56212; 56213; 56214; 56220; 56222; 56223;
56224; 56225; 56226; 14669; 56227; 56228; 56229; 56230; 7726; 2274;
56232; 13014; 13015; 56239; 56240; 26578; 56246; 56247; 56248;
56249; 56251; 56252; 56253; 56254; 56255; 56256; 15692; 56258;
56260; 56262; 56263; 56265; 56266; 56267; 56268; 17312; 17313;
56273; 13743; 13744; 13742; 56276; 56277; 56278; 56280; 56281;
56282; 56283; 56284; 56285; 10517; 56289; 56290; 56295; 56298;
14791; 56299; 56300; 687; 7844; 13071; 56310; 56311; 15721; 27388;
13793.
[0289] The following SEQ ID NOs correspond to the amino acid
sequences of lung-specific proteins identified using SBS that have
also been identified by mass spectrometry as described in Table
78A: 57229; 57232; 57233; 13591; 16384; 16385; 16412; 16413; 57242;
57243; 22218; 16451; 16459; 57275; 57284; 26647; 57286; 57288;
3284; 3285; 57290.
[0290] The following SEQ ID NOs correspond to the amino acid
sequences of lymph node-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 26363; 26364; 57462; 57463; 57464; 2250; 26503; 2256;
2258; 26504; 2260; 26505; 57467; 57468; 57469; 2262; 2266; 2267;
57474.
[0291] The following SEQ ID NOs correspond to the amino acid
sequences of lymphocyte-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 9528; 30736; 30737; 57636; 32496; 12212; 57645; 57646;
57650; 26351; 57651; 57652; 57653; 57669; 57671; 57676; 57681;
26448; 57686; 9984; 57687; 57696; 57697; 57699; 28867; 57703;
57723; 57724; 19601; 57733; 6541; 6542; 6544; 6545; 57738; 57739;
57740; 3297; 57745; 57747.
[0292] The following SEQ ID NOs correspond to the amino acid
sequences of monocyte-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 19373; 19374; 19375; 19376; 19377; 57651; 26356; 1369;
14621; 19535; 57949; 18175; 57951; 57952; 2271; 10294; 16541;
19608; 7756; 7757; 2306; 19612; 20394; 10559; 19720; 57979.
[0293] The following SEQ ID NOs correspond to the amino acid
sequences of muscle-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 2095; 2096; 2097; 1313; 9460; 58201; 58202; 58203;
58204; 58205; 58206; 14458; 14459; 14460; 14461; 58208; 9520; 9521;
9522; 9523; 22101; 58218; 58219; 58220; 14511; 14516; 14517; 58230;
1388; 1389; 14556; 14555; 58235; 58239; 58242; 14644; 58270; 58278;
58279; 14673; 14671; 14672; 10199; 22387; 22388; 22389; 22390;
31598; 10238; 31599; 31601; 31602; 14693; 3200; 14709; 14710;
58291; 58292; 58293; 58294; 58295; 58296; 58297; 14735; 58299;
2317; 14772; 58310; 3263; 58314; 58315; 58316; 10606; 58320; 58328;
1575; 1576; 18403; 14835; 3308; 3309; 3310; 3311; 3312; 1590.
[0294] The following SEQ ID NOs correspond to the amino acid
sequences of ovary-specific proteins identified using SBS that have
also been identified by mass spectrometry as described in Table
78A: 58747; 58748; 58749; 58751; 58753; 58755; 32284.
[0295] The following SEQ ID NOs correspond to the amino acid
sequences of pancreas-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 18875; 18874; 18876; 18873; 17978; 58866; 58868; 58869;
6011; 58871; 18881; 58880; 58881; 58882; 58883; 13654; 58907;
25068; 58917; 20386; 13717; 18895; 58922; 58923; 25081; 25080;
20395; 58925; 18901; 58928; 1540; 58932; 58933; 58935.
[0296] The following SEQ ID NOs correspond to the amino acid
sequences of prostate-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 13537; 22031; 22032; 17187; 22047; 4165; 15579; 59342;
59343; 59344; 59345; 59346; 59347; 20311; 22144; 22146; 22147;
59357; 55371; 55372; 55373; 22252; 22253; 22254; 22255; 22257;
22259; 1442; 1443; 6225; 17263; 6226; 59361; 59362; 59363; 59364;
59365; 59366; 59367; 20969; 24519; 22313; 22318; 22319; 22320;
22324; 22329; 22373; 22387; 22388; 22389; 22390; 30938; 22499;
22500; 59403; 59404; 59405; 59406; 59407; 22519; 22547; 22559;
59410.
[0297] The following SEQ ID NOs correspond to the amino acid
sequences of skin-specific proteins identified using SBS that have
also been identified by mass spectrometry as described in Table
78A: 59814; 59815; 59820; 32491; 32490; 59825; 24448; 24450; 30746;
59826; 59827; 59828; 59346; 59347; 59831; 59832; 59833; 59838;
59839; 59840; 59841; 59842; 59843; 466; 3055; 3056; 3057; 3058;
59845; 59847; 59851; 59852; 59856; 59357; 59870; 59871; 59872;
59873; 59874; 59875; 59878; 59881; 59882; 59886; 59887; 17262;
59888; 59889; 59890; 59891; 59892; 59893; 59894; 59895; 6225;
17263; 6226; 59896; 59897; 59900; 59912; 30817; 59914; 20963;
59362; 59363; 59364; 59365; 59924; 59925; 59951; 59958; 59965;
59967; 59968; 59969; 59973; 59974; 59977; 24540; 59978; 59979;
59981; 24545; 59984; 21042; 59987; 59988; 30938; 59989; 59991;
59992; 59403; 59405; 59997; 30960; 25597; 25598; 60005; 60007;
60012; 60013; 60014; 23916; 60016; 60017; 60018; 60025; 60026.
[0298] The following SEQ ID NOs correspond to the amino acid
sequences of small intestine-specific proteins identified using SBS
that have also been identified by mass spectrometry as described in
Table 78A: 12878; 60534; 20843; 60536; 60539; 24920; 20280; 15568;
60544; 60545; 4147; 60546; 24963; 60571; 60572; 60573; 60574;
60576; 60578; 60579; 25526; 25527; 60590; 60596; 13655; 60606;
60608; 2257; 57467; 2268; 56209; 56210; 60621; 60622; 60623; 60624;
13679; 60627; 15659; 60637; 60638; 60639; 25064; 60643; 60644;
25068; 24539; 13709; 60650; 25076; 25092; 13750; 60668; 12376;
60669; 16621; 13779; 60682; 7847; 13792; 27388; 13793.
[0299] The following SEQ ID NOs correspond to the amino acid
sequences of spleen-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 2136; 26350; 61144; 2177; 56093; 61145; 2227; 2228;
61149; 61150; 2252; 2253; 61157; 2259; 2261; 56213; 18267; 61163;
12323; 61167; 26673.
[0300] The following SEQ ID NOs correspond to the amino acid
sequences of stomach-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 61304; 61311; 61312; 16384; 16385; 61323; 61333; 61351;
61352; 61357; 27382.
[0301] The following SEQ ID NOs correspond to the amino acid
sequences of testes-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 62513; 28427; 62520; 9450; 9451; 9452; 9453; 9454; 9455;
9456; 9457; 9458; 62524; 5922; 5924; 62527; 28437; 28438; 28439;
62534; 9495; 28452; 62539; 2112; 62541; 22056; 28459; 3001; 28465;
13544; 13545; 9546; 28483; 28484; 62550; 62551; 62552; 19371;
62553; 62554; 28495; 24942; 62566; 28501; 28504; 62575; 62578;
62583; 28513; 62594; 62597; 62598; 12896; 28520; 9574; 62606;
28541; 28542; 28548; 4206; 62635; 62643; 62644; 20858; 28564;
28568; 28566; 28569; 28570; 28576; 28577; 62647; 3033; 28581;
20869; 1358; 16383; 23654; 62674; 28622; 28625; 28627; 29262;
30762; 62689; 62691; 28634; 62696; 62697; 62698; 62699; 62706;
28651; 28660; 62719; 59842; 62723; 28675; 28676; 62725; 9754;
62727; 6071; 1388; 1389; 62730; 4281; 4280; 4282; 62737; 2192;
62738; 9828; 28710; 28729; 62762; 62763; 18094; 18092; 18093;
28748; 28755; 28756; 28757; 28759; 28777; 62803; 19495; 28784;
62807; 62818; 62819; 6126; 62823; 62824; 3099; 28816; 62827; 62828;
28817; 16448; 32529; 32530; 28820; 28821; 15634; 62838; 9985;
14627; 22244; 28856; 28858; 62850; 28870; 62852; 62853; 62855;
3148; 62856; 62858; 28876; 62886; 62887; 28921; 28934; 28940;
28959; 28961; 62944; 62945; 22293; 63004; 63097; 63107; 63123;
63131; 63213; 63215; 63216; 63217; 63237; 29065; 32283; 63263;
1447; 29077; 26527; 29078; 63268; 63279; 29085; 15658; 29092;
63287; 63292; 29101; 63298; 26538; 63304; 29109; 63308; 29121;
63312; 63313; 13695; 13696; 3202; 3203; 4475; 63317; 6472; 10285;
10286; 29128; 2290; 2292; 63324; 63325; 63335; 63336; 29159; 1485;
63345; 29185; 10338; 63350; 63351; 29195; 63353; 16548; 16549;
7767; 29217; 63364; 29225; 29226; 29227; 14757; 14758; 10426;
22474; 639; 642; 10444; 4559; 63382; 29258; 63386; 63390; 29261;
28626; 29263; 63401; 63402; 1526; 63403; 25090; 52890; 18371;
22507; 22509; 22510; 673; 674; 675; 2357; 2358; 63419; 676; 63424;
29314; 63435; 29319; 3276; 3277; 6624; 63452; 63453; 63454; 18392;
63460; 29354; 63468; 29364; 13065; 63483; 63484; 63489; 713; 29376;
63496; 4649; 29387; 63497; 3314; 3315; 29388; 29391; 4655; 63518;
63520; 63521; 63529; 63531; 29423; 29424; 29425.
[0302] The following SEQ ID NOs correspond to the amino acid
sequences of trachea-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 31511; 65263; 65265; 31891; 28581; 65276; 12230; 18065;
2245; 65305; 2269; 16511; 16512; 31920; 65310; 59977; 31926; 65314;
25096; 10640; 31654; 22592.
[0303] The following SEQ ID NOs correspond to the amino acid
sequences of uterus-specific proteins identified using SBS that
have also been identified by mass spectrometry as described in
Table 78A: 28433; 65481; 32096.
[0304] The following SEQ ID NOs correspond to the amino acid
sequences of sex organ, prostate-specific proteins identified using
SBS that have also been identified by mass spectrometry as
described in Table 79A: 384; 1351; 59346; 59347; 65564; 65565;
14604; 65566; 65567; 65568; 65569; 65570; 4329; 59357; 6225; 17263;
6226; 59362; 59363; 59364; 59365; 65577; 65578; 65585; 65587;
65588; 65589; 14670; 30938; 65605; 65606; 4577; 59403; 59405;
22526.
[0305] The following SEQ ID NOs correspond to the amino acid
sequences of sex organ, testes-specific proteins identified using
SBS that have also been identified by mass spectrometry as
described in Table 79A: 65663; 65664; 65665; 28458; 65667; 401;
65670; 65671; 65672; 65673; 28581; 9651; 65678; 9746; 9747; 12315;
12316; 65694; 65695; 1511; 53801.
[0306] The following SEQ ID NOs correspond to the amino acid
sequences of sex organ, breast-specific proteins identified using
SBS that have also been identified by mass spectrometry as
described in Table 79A: 10336; 65694; 65695; 17312; 17313.
[0307] The following SEQ ID NO correspond to the amino acid
sequences of sex organ, cervix-specific proteins identified using
SBS that have also been identified by mass spectrometry as
described in Table 79A: 14670.
[0308] The following SEQ ID NOs correspond to the amino acid
sequences of sex organ, uterus-specific proteins identified using
SBS that have also been identified by mass spectrometry as
described in Table 79A: 65670; 65671; 65672; 65673; 65799.
[0309] SEQ ID NOs:32935-52639 correspond to amino acid sequences of
peptides previously identified by mass spectrometry that map to the
organ-specific proteins as described in Table 43B.
[0310] SEQ ID NOs:52640-52699 correspond to amino acid sequences of
peptides previously identified by mass spectrometry that map to the
organ-specific proteins as described in Table 44B.
[0311] SEQ ID NOs:52700-52864 correspond to amino acid sequences of
peptides previously identified by mass spectrometry that map to the
organ-specific proteins as described in Table 45B.
[0312] SEQ ID NOs:65803-72641 correspond to amino acid sequences of
peptides previously identified by mass spectrometry that map to the
organ-specific proteins as described in Table 78B.
[0313] SEQ ID NOs:72642-72688 correspond to amino acid sequences of
peptides previously identified by mass spectrometry that map to the
organ-specific proteins as described in Table 79B.
DETAILED DESCRIPTION OF THE INVENTION
[0314] The present invention relates generally to organ-specific
proteins and polynucleotides that encode them. In particular the
invention relates to diagnostic panels comprising reagents to
detect organ-specific proteins or polynucleotides and methods of
identifying and using the same.
[0315] Because the blood bathes all of the organs of the body, the
blood contains, as noted above, proteins that are secreted, leaked,
excreted or shed from the cells of all the organs in the body.
These proteins can provide information about the organs and serve
as reporter groups or markers that accurately reflect the health or
disease state of each organ or groups of organs. This is because
under ordinary conditions the levels of these organ-specific
proteins secreted or shed into the blood may attain normal levels,
whereas under disease conditions the levels of the proteins may
change, reflecting the altered behavior (e.g., control of protein
expression) of the disease-perturbed networks in the disease organ.
Thus the levels or organ-specific proteins in the blood will be
altered with health and disease and, indeed, may be specifically
altered for each type of disease for a particular organ and each
stage of progression for each disease. Highly sensitive
blood-protein diagnostics of organ-specific fingerprints could be
used to detect the early stages of disease and monitor treatment
when therapeutic intervention is most effective. Specific proteins
in blood may be used as markers to diagnose disease at the earliest
stages. Expression array studies have shown that such proteins, or
protein panels, exist in cells and can serve as markers of disease
progression or disease prognosis (E. E. Schadt et al., Nat Genet
(2005) 37: 710, H. Dai et al., Cancer Res (2005) 65:4059). However,
specifically identifying such proteins has proved difficult.
Particularly, when attempting to detect those proteins that are
tissue or organ specific as well as secreted.
[0316] A systems view of disease is predicated upon a very simple
idea--that disease arises from biological networks that have been
disease perturbed either by gene mutations or pathogenic
environmental signals (e.g., infections). These perturbed networks
alter the expression levels of proteins they encode and these lead
to the pathological symptoms of disease. Furthermore, a fraction of
these proteins are expressed only by the organ of interest (are
organ-specific) and it is postulated are secreted (or shed or
deposited after cell destruction, etc) into the blood with distinct
levels that correlate with health and each type of disease
occurring in the organ. Thus each human organ or tissue type has a
unique molecular fingerprint in the blood comprising distinct
levels of organ-specific proteins. Blood, itself, may be considered
an organ that circulates throughout the body and is in contact with
all other organs and the protein concentrations or the
organ-specific fingerprints serve as a diagnostic vehicle to
measure the state of health or disease of a subject. Although,
blood is a medium to measure the state of health and disease,
significant limitations exist with current diagnostic assays that
delay or prevent early diagnosis when it would be most
effective.
[0317] Early diagnosis of disease by measuring changes in proteins
in the blood would lead to earlier treatment and therefore
healthier outcomes for patients. The determination of predetermined
normal ranges of low abundance proteins in healthy organs gives
diagnosticians a crucial advantage in health care: the potential to
define disease at the earliest stages and initiate treatment when
it may be most effective. If the organ-specific proteins that are
normally found within a healthy organ could be identified and
measured, the diagnostician would have the distinct advantage of
comparing a patient sample to a set of expected normal values of
blood proteins that are typically found in a state of health in an
organ.
[0318] This invention pre-defines normal organ-specific protein
sets specifically identified and quantified for each of multiple
healthy human organs and major tissue types. These organ-specific
proteins identified from healthy human organs may, in whole or in
part, be used as markers or identifiers for health and disease. The
levels of these organ-specific proteins in blood from diseased
individuals may be distinguished from the levels of these
organ-specific proteins in the blood of healthy individuals. By
identifying organ-specific protein markers and measuring the level
of these proteins in normal blood, the status of health or disease
may be monitored through the correlation of the levels of proteins
in this organ-specific fingerprint at the earliest stages of
disease and lead to early diagnosis and treatment.
[0319] Thus, the present invention provides organ-specific proteins
that serve as markers to measure changes in the status of an organ
or organs to measure health and diagnose disease. The inventive
markers, obtained from normal, healthy organ tissue (see Tables
1-32, 36-45 and 47-79) are used as a library of biological
indicators to identify organ-specific blood proteins that are
secreted, leaked, excreted or shed into blood in a human or mammal.
Such markers can be used individually or collectively. For example
a single marker for an organ or tissue could be used to monitor
that organ or tissue. However, adding additional markers from that
tissue to the assay will improve the diagnostic power as well as
the sensitivity of the assay. Further, one of skill in the art can
readily appreciate that probes to such markers, be they nucleic
acid probes, nanoparticles, or polypeptides (e.g., antibodies) can
comprise a kit, lateral flow test kit or an array and can include a
few probes to proteins from several organs or several probes to
proteins from one organ or tissue. For example, in one kit or assay
device a whole body health assay may be used wherein several
markers are tracked for every organ and when one or more organ or
tissue demonstrates a deviation from normal a more rigorous test is
performed with many more markers for that organ or tissue.
Likewise, entire organ set assays may be devised. In such an
example a cardiovascular assay may be employed wherein
tissue/organ-specific markers from heart and lung are the basis of
the assay kit.
[0320] One of skill in the art can readily appreciate that the
application of these marker sets that are tissue and organ-specific
are virtually limitless. From using as diagositic and prognostic
indicators, to use in following drug treatment or in drug discovery
to determine what proteins and genes are affected. Further, such
markers can easily be used in combination with antibodies for other
ligands for drug targeting or imaging via MRI or PET or by other
means. In such examples, a marker specific for prostate could form
the basis for targeted cancer therapy or possible imaging/therapy
of metastatic cancer derived from prostate. The comparison of the
normal levels of organ-specific proteins to the levels of these
proteins found in a sample of patient blood or bodily fluid or
other biological sample, such as a biopsy can be used to define
normal health, detect the early stages of disease, monitor
treatment, prognosticate disease, measure drug responses, titrate
administered drug doses, evaluate efficacy, stratify patients
according to disease type (e.g., prostate cancer may well have four
or more major types) and define therapeutic targets when
therapeutic intervention is most effective. This invention provides
pre-defined normal organ-specific proteins and protein sets that
have been specifically identified and quantified for each of 32 or
more healthy, human organs examined. These organ-specific proteins
identified from healthy, human organs may be used as markers or
identifiers for health and disease and/or may be distinguished from
constitutive proteins in the blood, fluid, or tissue. By using the
approach of comparing the proteins found in a sample of blood with
the organ-specific protein markers that have been identified as
specific to a healthy organ, the status of health or disease may be
monitored at the earliest stage and lead to early diagnosis and
treatment.
[0321] When there is a change in health status that affects an
organ, the blood fingerprint that is measured is reflective of the
particular target organ. Proteins that comprise the organ-specific
blood fingerprint will either increase or decrease in level in
response to the changes brought by the stimulus of the disease. The
increase or decrease in the amount in blood (or components specific
for a cell, tissue or organ) of the components of the
organ-specific blood fingerprint may be quantified by antibodies
(or other specific protein-capture agents) specific for the
proteins, by proteomic techniques (e.g., mass spectrometry) or by
measurement with microfluidic and/or nanotechnology sensors and
compared to the normal level of the organ-specific proteins. The
disease-perturbed networks may alter the expression patterns of
virtually any different type of proteins--those involved in signal
transduction pathways, those involved in the execution of cellular
differentiation, those involved in the response to physiological
stimuli, those involved in the normal cellular functions such as
the cell cycle, etc, and those involved in mediating whom cells
will interact with or where they will migrate. When disease strikes
an organ, the physical response may, for example, involve changes
in the proteins that connect together in biological signal
transduction networks to send information to other protein effector
proteins also altering their levels of expression. These signal
transduction pathways communicate changes in the body in response
to a stimulus or disease. These signal transduction pathways also
serve as a response network to a stimulus or disease. An example of
a response network to a disease is the inflammatory pathway
mediated by Phospholipase A2 (PLA2). PLA2 is modulated and may be
used as a marker in the diagnosis of cardiovascular disease
(Sudhir, K., J Clin Endocrinol Metab (2005) 90:3100-5),
arteriosclerosis (Smitzko, et al., Circulation (2003),
108:2041-2048; Sunara et al., Cell Mol Life Sci (2005)
62:2487-2494)), neurodegenerative disease (Farooqui et al.,
Neurchem Res, (2004), 11:1961-1977), allergic disease (Triggiani et
al., Journal of Allergy and Clinical Immunology, (2005)
116:1000-1006). Another example of effector protein changes that
may be measured by blood fingerprints is the regulation of map
kinase in response to cardiovascular disease or in certain cancers
or tumors, including prostate cancer (Kopper et al., Pathology of
Oncology Research (2005), 11:197203). Changes in signaling proteins
serve as biological markers or blood fingerprints that may be used
to diagnose or monitor disease.
[0322] As one of skill in the art can readily appreciate, certain
aspects of the present invention refer to known protein and nucleic
acid sequences. Wherein such sequences are included in a diagnostic
or prognostic panel and have previously been described as
indicative of disease or perturbation of that organ the inventive
panel should comprise at least one additional organ-specific marker
(nucleic acid or polypeptide sequence or detection reagent
thereto). Accordingly, wherein a known sequence, either nucleic
acid or polypeptide sequence, is included in a panel or mixture and
wherein said sequence has been demonstrated by the art to be
previously associated with the particular organ and/or indicative
of perturbation such sequences should also be associated with at
least one sequence not previously specifically associated with the
organ and/or disease/perturbation.
[0323] Prior to setting forth the invention in further detail, it
may be helpful to an understanding thereof to set forth definitions
of certain terms that will be used hereinafter.
[0324] The term "blood" refers to whole blood, plasma or serum
obtained from a mammal.
[0325] In the practice of the invention, an "individual" or
"subject" refers to vertebrates, particularly members of a
mammalian species, and includes, but is not limited to, primates,
including human and non-human primates, domestic animals, and
sports animals.
[0326] "Component" or "member" of a set refers to an individual
constituent protein, peptide, nucleotide or polynucleotide of an
organ-specific set.
[0327] As used herein an "organ-specific protein set" is made up of
the set of organ-specific proteins identified from an organ sample
obtained from a normal, healthy individual using the methods
described herein (see, e.g., Example 1 and Example 9). Illustrative
organ-specific protein sets are provided in Tables 1-32, 36-45 and
47-79 and were identified using analysis of MPSS transcripts as
described further herein and using sequencing by synthesis (SBS)
analysis as described further herein. Individual proteins that make
up the set are referred to herein as components or members of the
set. In the examples and recitation below, blood is used as the
prototypic example, however, it should be understood that any
biological fluid or sample may be exchanged for the terms blood,
serum, or plasma. Accordingly, normal organ-specific blood
fingerprint can be exchanged with "organ-specific
saliva/urine/tissue, etc. fingerprint".
[0328] As used herein, a "normal serum organ-specific protein set"
comprises the subset of proteins from an organ-specific protein set
that are detected in normal serum. Individual proteins that make up
the set are referred to herein as components or members of the
set.
[0329] As used herein, a "normal organ-specific blood fingerprint"
is a data set comprising the determined levels in blood from
normal, healthy individuals of one, two, three, four, five, six,
seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,
fifteen, sixteen, seventeen, eighteen, nineteen, twenty,
twenty-one, twenty-two, twenty-three, twenty-four, twenty-five,
twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty,
thirty-one, thirty-two, thirty-three, thirty-four, thirty-five,
thirty-six, thirty-seven, thirty-eight, thirty-nine, forty,
forty-one, forty-two, forty-three, forty-four, forty-five,
forty-six, forty-seven, forty-eight, forty-nine, fifty, sixty,
seventy, eighty, ninety, one-hundred or more components of a serum
organ-specific protein set of one organ, but could comprise
multiples thereof if more than one organ is analyzed. The normal
levels in the blood for each component included in a fingerprint
are determined by measuring the level of protein in the blood using
any of a variety of techniques known in the art and described
herein, in a sufficient number of blood samples from normal,
healthy individuals to determine the standard deviation (SD) with
statistically meaningful accuracy. Thus, as would be recognized by
one of skill in the art, a determined normal level is defined by
averaging the level of protein measured in a statistically large
number of blood samples from normal, healthy individuals and
thereby defining a statistical range of normal. A normal
organ-specific blood fingerprint comprises the determined levels in
normal, healthy blood of N members of a serum organ-specific
protein set wherein N is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, or more members up to the total number of members in a
given serum organ-specific protein set per organ being profiled. In
certain embodiments, a normal organ-specific blood fingerprint
comprises the determined levels in normal, healthy blood of at
least two components of a serum organ-specific protein set. In
other embodiments, a normal organ-specific blood fingerprint
comprises the determined levels in normal, healthy blood of at
least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 components of a serum organ-specific protein set. In yet
further embodiments, a normal organ-specific blood fingerprint
comprises the presence or absence of organ, cell or tissue-specific
proteins or transcripts and may or may not rely on absolute levels
of said components per se. In specific embodiments, merely a change
over a baseline measurement for a particular individual may be
used. In such an embodiment, levels or mere presence or absence of
proteins or transcripts from blood, body fluid or tissue may be
measured at one time point and then compared to a subsequent
measurement, hours, days, months or years later. Accordingly,
normal changes per individual can be zeroed out and only those
proteins or transcripts that change over time are focused on.
[0330] As used herein, a "predetermined normal level" is an average
of the levels of a given component measured in a statistically
large number of blood samples from normal, healthy individuals.
Thus, a predetermined normal level is a statistical range of normal
and is also referred to herein as "predetermined normal range". The
normal levels or range of levels in the blood for each component
are determined by measuring the level of protein in the blood using
any of a variety of techniques known in the art and described
herein in a sufficient number of blood samples from normal, healthy
individuals to determine the standard deviation (SD) with
statistically meaningful accuracy. In one embodiment it may be
useful to determine average levels for individual falling into
different age groups (e.g. 1-2, 3-5, 6-8, 9-12 and so forth if,
indeed, these levels change with age). In another embodiment, one
may also want to determine the levels at certain times of the day,
at certain times from having eaten a meal, etc. One may also
determine how common physiological stimuli affect the
organ-specific blood fingerprints.
[0331] As used herein a "disease-associated organ-specific blood
fingerprint" is a data set comprising the determined level in a
blood sample from an individual afflicted with a disease of one or
more components of a normal serum organ-specific protein set that
demonstrates a statistically significant change as compared to the
determined normal level (e.g., wherein the level in the disease
sample is above or below a predetermined normal range). The data
set is compiled from samples from individuals who are determined to
have a particular disease using established medical diagnostics for
the particular disease. The blood (serum) level of each protein
member of a normal serum organ-specific protein set as measured in
the blood of the diseased sample is compared to the corresponding
determined normal level. A statistically significant variation from
the determined normal level for one or more members of the normal
serum organ-specific protein set provides diagnostically useful
information (disease-associated fingerprint) for that disease.
Thus, note that it may be determined for a particular disease or
disease state that the level of only a few members of the normal
serum organ-specific protein set change relative to the normal
levels. Thus, a disease-associated organ-specific blood fingerprint
may comprise the determined levels in the blood of only a subset of
the components of a normal serum organ-specific protein set for a
given organ and a particular disease. Thus, a disease-associated
organ-specific blood fingerprint comprises the determined levels in
blood (or as noted herein any bodily fluid or tissue sample,
however in most embodiments samples from blood are compared with a
normal from blood and so on) of N members of a serum organ-specific
protein set wherein N is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 60, 70, 80, 90, 100, 110 or more or any integer value
therebetween, or more members up to the total number of members in
a given serum organ-specific protein set. In this regard, in
certain embodiments, a disease-associated organ-specific blood
fingerprint comprises the determined levels of one or more
components of a normal serum organ-specific protein set. In one
embodiment, a disease-associated organ-specific blood fingerprint
comprises the determined levels of at least two components of a
normal serum organ-specific protein set. In other embodiments, a
disease-associated organ-specific blood fingerprint comprises the
determined levels of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110 or more or any integer
value therebetween components of a normal serum organ-specific
protein set.
[0332] The term "test compound" refers in general to a compound to
which a test cell is exposed, about which one desires to collect
data. Typical test compounds will be small organic molecules,
typically prospective pharmaceutical lead compounds, but can
include proteins (e.g., antibodies), peptides, polynucleotides,
heterologous genes (in expression systems), plasmids,
polynucleotide analogs, peptide analogs, lipids, carbohydrates,
viruses, phage, parasites, and the like.
[0333] The term "biological activity" as used herein refers to the
ability of a test compound to alter the expression of one or more
genes or proteins.
[0334] The term "test cell" refers to a biological system or a
model of a biological system capable of reacting to the presence of
a test compound, typically a eukaryotic cell or tissue sample, or a
prokaryotic organism.
[0335] The term "gene expression profile" refers to a
representation of the expression level of a plurality of genes in
response to a selected expression condition (for example,
incubation in the presence of a standard compound or test
compound). Gene expression profiles can be expressed in terms of an
absolute quantity of mRNA transcribed for each gene, as a ratio of
mRNA transcribed in a test cell as compared with a control cell,
and the like or the mere presence or absence of a protein an RNA
transcript or more generally gene expression. As used herein, a
"standard" gene expression profile refers to a profile already
present in the primary database (for example, a profile obtained by
incubation of a test cell with a standard compound, such as a drug
of known activity), while a "test" gene expression profile refers
to a profile generated under the conditions being investigated. The
term "modulated" refers to an alteration in the expression level
(induction or repression) to a measurable or detectable degree, as
compared to a pre-established standard (for example, the expression
level of a selected tissue or cell type at a selected phase under
selected conditions).
[0336] "Similar", as used herein, refers to a degree of difference
between two quantities that is within a preselected threshold. The
similarity of two profiles can be defined in a number of different
ways, for example in terms of the number of identical genes
affected, the degree to which each gene is affected, and the like.
Several different measures of similarity, or methods of scoring
similarity, can be made available to the user: for example, one
measure of similarity considers each gene that is induced (or
repressed) past a threshold level, and increases the score for each
gene in which both profiles indicate induction (or repression) of
that gene.
[0337] As used herein, the term "target specific" is intended to
mean an agent that binds to a target analyte selectively. This
agent will bind with preferential affinity toward the target while
showing little to no detectable cross-reactivity toward other
molecules. For example, when the target is a nucleic acid, a target
specific sequence is one that is complementary to the sequence of
the target and able to hybridize to the target sequence with little
to no detectable cross-reactivity with other nucleic acid
molecules. A nucleic acid target could also be bound in a target
specific manner by a protein, for example by the DNA binding domain
of a transcription factor. If the target is a protein or peptide it
can be bound specifically by a nucleic acid aptamer, or another
protein or peptide, or by an antibody or antibody fragment which
are sub-classes of proteins.
[0338] As used herein, the term "genedigit" is intended to mean a
region of pre-determined nucleotide or amino acid sequence that
serves as an attachment point for a label. The genedigit can have
any structure including, for example, a single unique sequence or a
sequence containing repeated core elements. Each genedigit has a
unique sequence which differentiates it from other genedigits. An
"anti-genedigit" is a nucleotide or amino acid sequence or
structure that binds specifically to the gene digit. For example,
if the genedigit is a nucleic acid, the anti-genedigit can be a
nucleic acid sequence that is complementary to the genedigit
sequence. If the genedigit is a nucleic acid that contains repeated
core elements then the anti-genedigit can be a series of repeat
sequences that are complementary to the repeat sequences in the
genedigit. An anti-genedigit can contain the same number, or a
lesser number, of repeat sequences compared to the genedigit as
long as the anti-genedigit is able to specifically bind to the
genedigit.
[0339] As used herein, the term "specifier" is intended to mean the
linkage of one or more genedigits to a target specific sequence.
The genedigits can be directly linked or can be attached using an
intervening or adapting sequence. A specifier can contain a target
specific sequence which will allow it to bind to a target analyate.
An "anti-specifier" has a complementary sequence to all or part of
the specifier such that it specifically binds to the specifier.
[0340] As used herein, the term "label" is intended to mean a
molecule or molecules that render an analyte detectable by an
analytical method. An appropriate label depends on the particular
assay format and are well known by those skilled in the art. For
example, a label specific for a nucleic acid molecule can be a
complementary nucleic acid molecule attached to a label monomer or
measurable moiety, such as a radioisotope, fluorochrome, dye,
enzyme, nanoparticle, chemiluminescent marker, biotin, or other
moiety known in the art that is measurable by analytical methods.
In addition, a label can include any combination of label
monomers.
[0341] As used herein, "unique" when used in reference to label is
intended to mean a label that has a detectable signal that
distinguishes it from other labels in the same mixture. Therefore,
a unique label is a relative term since it is dependent upon the
other labels that are present in the mixture and the sensitivity of
the detection equipment that is used. In the case of a fluorescent
label, a unique label is a label that has spectral properties that
significantly differentiate it from other fluorescent labels in the
same mixture. For example, a fluorescein label can be a unique
label if it is included in a mixture that contains a rhodamine
label since these fluorescent labels emit light at distinct,
essentially non-overlapping wavelengths. However, if another
fluorescent label was added to the mixture that emitted light at
the same or very similar wavelength to fluorescein, for example the
Oregon Green fluorophore, then the fluorescein would no longer be a
unique label since Oregon Green and fluorescein could not be
distinguished from each other. A unique label is also relative to
the sensitivity of the detection equipment used. For example, a
FACS machine can be used to detect the emission peaks from
different fluorophore-containing labels. If a particular set of
labels have emission peaks that are separated by, for example, 2 nm
these labels would not be unique if detected on a FACS machine that
can distinguish peaks that are separated by 10 nm or greater, but
these labels would be unique if detected on a FACS machine that can
distinguish peaks separated by 1 nm or greater.
[0342] As used herein, the term "signal" is intended to mean a
detectable, physical quantity or impulse by which information on
the presence of an analyte can be determined. Therefore, a signal
is the read-out or measurable component of detection. A signal
includes, for example, fluorescence, luminescence, calorimetric,
density, image, sound, voltage, current, magnetic field and mass.
Therefore, the term "unit signal" as used herein is intended to
mean a specified quantity of a signal in terms of which the
magnitudes of other quantities of signals of the same kind can be
stated. Detection equipment can count signals of the same type and
display the amount of signal in terms of a common unit. For
example, a nucleic acid can be radioactively labeled at one
nucleotide position and another nucleic acid can be radioactively
labeled at three nucleotide positions. The radioactive particles
emitted by each nucleic acid can be detected and quantified, for
example in a scintillation counter, and displayed as the number of
counts per minute (cpm). The nucleic acid labeled at three
positions will emit about three times the number of radioactive
particles as the nucleic acid labeled at one position and hence
about three times the number of cpms will be recorded.
[0343] Because the disease-perturbed networks in the organ may
initiate the expression of one or more proteins whose synthesis it
does not ordinarily control, it should be noted that, in certain
embodiments, a disease-associated organ-specific blood fingerprint
will comprise the determined level of one or more components of a
normal organ-specific protein set that are NOT components of the
corresponding normal serum organ-specific protein set. Thus, in
this regard, a disease-associated organ-specific blood fingerprint
may comprise the determined level of one or more components of a
normal organ-specific protein set or may comprise a protein or set
of proteins not detected in a normal organ-specific protein set.
Further, in certain embodiments, a disease-associated
"organ-specific" blood fingerprint comprises the determined levels
of one or more components of one, two, three, four, five, six,
seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90,
100, 110 or any integer value therebetween or more normal serum
organ-specific protein sets. Further, in additional embodiments,
the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
60, 70, 80, 90, 100, 110 or more or any integer value therebetween
components of multiple sets could be combined for analysis of
multiple organs, tissues, systems, or cells. Thus, in this regard,
a disease-associated organ-specific blood fingerprint may comprise
the determined levels of one or more components from 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110 or
any integer value therebetween components or more normal serum
organ-specific protein sets.
[0344] The term "polynucleotide" refers to a polymeric form of
nucleotides of any length, including deoxyribonucleotides or
ribonucleotides, which can comprise analogs thereof.
[0345] As used herein, "purified" refers to a specific protein,
polypeptide, or peptide composition that has been subjected to
fractionation to remove various other proteins, polypeptides, or
peptides, and which composition substantially retains its activity,
as may be assessed, for example, by any of a variety of protein
assays known to the skilled artisan for the specific or desired
protein, polypeptide or peptide.
[0346] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The terms also encompass an amino acid polymer that has
been modified; for example, by disulfide bond formation,
glycosylation, lipidation, or conjugation with a labeling
component.
[0347] The terms "glycopeptide" or "glycoprotein" refers to a
peptide that contains covalently bound carbohydrate. The
carbohydrate can be a monosaccharide, oligosaccharide or
polysaccharide.
Organ-Specific Protein Sets
[0348] The invention provides organ-specific protein sets. An
organ-specific protein set is made up of the set of organ-specific
proteins (as defined further herein) identified from a normal,
healthy sample of a particular organ using the methods described
herein (see, e.g., Example 1 and Example 9). Illustrative
organ-specific protein sets include those provided in Tables 1-32,
36-45 and 47-79. Amino acid and polynucleotide sequences for
illustrative organ-specific proteins are set forth in SEQ ID
NOs:1-72,689.
[0349] As used herein, the term "organ" is defined as would be
understood in the art. Thus, the term, "organ-specific" as used
herein generally refers to proteins (or transcripts) that are
primarily expressed in a single organ. In addition, in a complex
organ such as the brain, there will be distinct functional
subregions (e.g. the cortex, the cerebellum, the thalamus, etc)
that will be equivalent to different organs as defined above. It
should be noted that the skilled artisan would readily appreciate
upon reading the instant specification that cell-specific
transcripts and proteins and tissue-specific transcripts and
proteins are also contemplated in the present invention. Further,
as those of skill in the art would appreciate the transcriptomes
(e.g. quantitative collection of the full complement of mRNAs, or
transcripts in a particular tissue or organ at a particular time)
of organs that are specific for males or females should not be
included when assessing organ-specific transcripts (or proteins) of
the opposite sex (in this regard exemplary tables and analysis are
set forth in Tables 36-42, 44, 45, 72-77 and 79). As such, and as
discussed further herein, in certain embodiments, organ-specific
protein is defined as a protein encoded by a transcript that is
expressed at a level of at least 3 copies/million (as measured, for
example, by massively parallel signature sequencing (MPSS) or
sequencing by synthesis (SBS)) in the cell/tissue/organ of interest
but is expressed at less than 3 copies/million in other
cells/tissues/organs. In a further embodiment, an organ-specific
protein is one that is encoded by a transcript that is expressed
95% in one organ and the remaining 5% in one or more other organs.
(In this context, total expression across all organs examined is
taken as 100%). In certain embodiments, an organ-specific protein
is one that is encoded by a transcript that is expressed at about
50%, 55%, 60%, 65%, 70%, 75%, 80% to about 90% in one organ and
wherein the remaining 10%-50% is expressed in one or more other
organs.
[0350] In one embodiment, organ-specific transcripts and proteins
encoded thereby are identified as follows:
[0351] Assume the expression (in tpm) and the associated SD of a
MPSS sequence tag in a tissue is {(X.sub.i, .sigma..sub.i)}, where
i=1, 2, . . . , 32 represents individual tissues. Assume the tag
has the highest expression levels in tissue m where the expression
and the SD are (X.sub.m, .sigma..sub.m). Three rules are then
applied to determine whether the tag is specific to tissue m as
follows:
[0352] i) The expression of the tag in tissue m is above a minimal,
estimated noise levels, i.e.,
X.sub.m.gtoreq.5. (1)
[0353] ii) The expression of the tag in tissue m is well above the
expression of the tag in all other tissues. More specifically, the
mean expression of the tag is first calculated in the other tissues
being examined (e.g., all tissues except tissue m) as
X _ = 1 N i .noteq. m X i , ( 2 ) ##EQU00001##
[0354] the associated standard error as
.sigma. X _ = 1 N i .noteq. m .sigma. i 2 , ( 3 ) ##EQU00002##
[0355] and the corresponding SD as
s = 1 N - 1 i .noteq. m ( X i - X _ ) 2 + 1 N i .noteq. m .sigma. i
2 , ( 4 ) ##EQU00003##
[0356] where N=31.
[0357] The significance that the expression of the tag in tissue m
is above the expression of the tag in other tissues is then
evaluated as
p dis = 1 2 erfc ( X m - X _ 2 ( s 2 + .sigma. m 2 + .sigma. X _ 2
) ) . ( 5 ) ##EQU00004##
[0358] For the tag to be specific to tissue m, in this embodiment,
it is required that
p.sub.dis.ltoreq.10.sup.-3. (6)
[0359] iii) The specificity f of the tag in tissue m has to be well
above a pre-selected cutoff value f.sub.0. More precisely, the
specificity of the tag in tissue m is defined as
f = X m i X i , ( 7 ) ##EQU00005##
[0360] and the associated SD is evaluated as
.sigma. f = f X m ( 1 - f ) 2 .sigma. m 2 + f 2 i .noteq. m .sigma.
i 2 . ( 8 ) ##EQU00006##
[0361] The significance that f was above f.sub.0 is then given
by
p spc = 1 2 erfc ( f - f 0 2 .sigma. f ) . ( 9 ) ##EQU00007##
[0362] In this embodiment, nine different values of f.sub.0 and
p.sub.spc can be applied in determining organ-specific MPSS tags,
ranging from the most stringent condition (f.sub.0=1 and
p.sub.spc.ltoreq.10.sup.-3) to the least stringent condition
(f.sub.0=0.5 and p.sub.spc.ltoreq.0.1). In one particular
embodiment, it is required that
p.sub.spc.ltoreq.10.sup.-3. (10)
[0363] The number of organ-specific tags varies with the selected
values of f.sub.0 and p.sub.spc.
[0364] As would be readily recognized by the skilled artisan upon
reading the present disclosure, in certain embodiments, an
organ-specific blood fingerprint can readily be discerned even if
some expression of an "organ-specific" protein from a particular
organ is detected at some level in another organ, or even more than
one organ. For example, the organ-specific blood fingerprint from
prostate can conclusively identify a particular prostate disease
(and stage of disease) despite expression of one or more protein
members of the fingerprint in one or more other organs. Thus, an
organ-specific protein as described herein may be predominantly or
differentially expressed in an organ of interest rather than
uniquely or specifically expressed in the organ. In this regard, in
certain embodiments, differentially expressed means at least 1.5
fold expression in the organ of interest as compared to other
organs. In another embodiment, differentially expressed means at
least 2 fold expression in the organ of interest as compared to
expression in other organs. In yet a further embodiment,
differentially expressed means at least 2.5, 3, 3.5, 4, 4.5, 5 fold
or higher expression in the organ of interest as compared to
expression of the protein in other organs. As described elsewhere
herein, "protein" expression can be determined by analysis of
transcript expression using a variety of methods.
[0365] In one embodiment, the organ-specific proteins are
identified by preparing RNA and/or a cDNA library from an organ,
tissue or biological fluid (e.g., whole blood, serum, etc.) of
interest. Any organ of a mammalian body is contemplated herein.
Illustrative organs include, but are not limited to, heart, kidney,
ureter, bladder, urethra, liver, prostate, heart, blood vessels,
bone marrow, skeletal muscle, smooth muscle, brain (amygdala,
caudate nucleus, cerebellum, corpus callosum, fetal, hypothalamus,
thalamus), spinal cord, peripheral nerves, retina, nose, trachea,
lungs, mouth, salivary gland, esophagus, stomach, small intestines,
large intestines, hypothalamus, pituitary, thyroid, pancreas,
adrenal glands, ovaries, oviducts, uterus, placenta, vagina,
mammary glands, testes, seminal vesicles, penis, lymph nodes, PBMC,
thymus, and spleen. As noted above, upon reading the present
disclosure, the skilled artisan would recognize that cell-specific
and tissue-specific proteins are contemplated herein and thus,
proteins specifically expressed in cells or tissues that make up
such organs are also contemplated herein. In certain embodiments,
in each of these organs, transcriptomes are obtained for the cell
types in which the disease of interest arises. For example, in the
prostate there are two dominant types of cells--epithelial cells
and stromal cells. About 98% of prostate cancers arise in
epithelial cells. Similarly, in the breast, 90% of cancers arise in
epithelial cells. As such, in certain embodiments, transcriptomes
are isolated from these particular cell types from an organ of
interest (e.g., prostate epithelial cells; breast epithelial
cells). In this regard, any cell type that makes up any of the
organs described herein is contemplated herein. Illustrative cell
types include, but are not limited to, epithelial cells, stromal
cells, cortical cells, endothelial cells, endodermal cells,
ectodermal cells, mesodermal cells, lymphocytes (e.g., B cells and
T cells including CD4+ T helper 1 or T helper 2 type cells, CD8+
cytotoxic T cells), all of the major types of white blood cells
present in the blood (e.g., eosinophils, megakaryoctyes,
granulocytes, macrophages, neutrophils, etc) erythrocytes,
keratinocytes, and fibroblasts. In the case of the white blood
cells, the organ-specific proteins can be obtain directly from the
isolated cell types and will not have to be secreted into the blood
for identification. Thus the organ-specific strategy will allow us
to assess any diseases of the white blood cell types (e.g.
neutrophils, basophils, eosinophils, macrophage, monocytes, and
lymphocytes (including B and T-lymphocytes). Particular cell types
within organs or tissues may be obtained by histological
dissection, by the use of specific cell lines (e.g., prostate
epithelial cell lines), by cell sorting or by a variety of other
techniques known in the art. Not only are the above parameters
useful in identifying organ-specific proteins or transcripts, but
such analysis can be used in harvesting mRNA and cDNA from a fluid,
tissue, organ of interest or blood for analysis.
[0366] In one embodiment, transcriptomes from a particular cell
type of an organ of interest (such as prostate epithelial cells,
breast epithelial cells, etc.) are isolated and analyzed using
methods as described herein to determine which transcripts are
organ-specific. The organ-specific transcripts identified from the
particular cell type of the organ can then be compared to the
organ-specific transcripts identified from whole organ samples
(e.g., the organ-specific proteins provided in Tables 1-32, 36-45
and 47-79) to determine those transcripts that overlap or to
identify additional organ-specific transcripts that may not have
been detected from the whole tissue due to sensitivity issues. In
this way, additional normal organ-specific protein members of a set
can be identified. Further, in certain embodiments, a subset of
normal organ-specific proteins can also be identified. For example,
a normal prostate-epithelial cell-specific protein subset can be
identified that is the set of proteins that are specifically
expressed in prostate-epithelial cells. Thus, particular cell types
from organs may include, but are not limited to, renal cortical
epithelial cells, hepatocytes, mammary epithelial cells, prostate
epithelial cells, renal proximal tubule epithelial cells, and
epidermal keratinocytes. This list is only exemplary and not meant
to be limiting.
[0367] As one of skill in the art can appreciate, technology in the
area of detection techniques is rapidly evolving. In particular,
techniques that only a few years ago required milligram quantities
of sample can now be performed with pictogram quantities.
Nanotechnology techniques can now be employed to assist in
detection of nucleic acid and polypeptide targets of the present
invention. Further, as this technology develops it will be feasible
to achieve single cell-specific transcripts. These single-cell
techniques are now available for abundant transcripts and can be
adapted by the skilled artisan to permit the analyses of low
abundance transcripts at the single cell level.
[0368] It should be noted that in certain embodiments,
organ-specific blood fingerprints can be determined from
"organ-specific" proteins from multiple organs, such as from organs
that share a common function or make up a system (e.g., digestive
system, circulatory system, respiratory system, cardiovascular
system, the immune system (including the different cells of the
immune system, such as, but not limited to, B cells, T cells
including CD4+ T helper 1 or T helper 2 type cells, regulatory T
cells, CD8+ cytotoxic T cells, NK cells, dendritic cells,
macrophages, monocytes, neutrophils, granulocytes, mast cells,
etc.), the sensory system, the skin, brain and the nervous system,
and the like). Accordingly, panels of probes to the organ-specific
components described herein can be fashioned in a way to analyze
multiple organ combinations.
Nucleic Acid Analysis
[0369] As noted above, in addition to detection of polypeptides
that are organ/tissue specific either in blood, tissue sample or
biological fluid, nucleic acid detection techniques offer
additional advantages due to sensitivity of detection. RNA can be
collected and/or generated from blood, biological fluids, tissues,
organs, cell lines, or other relevant sample using techniques known
in the art, such as those described in Kingston. (2002 Current
Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John
Wiley & Sons, Inc., NY, N.Y. (see, e.g., as described by Nelson
et al. Proc Natl Acad Sci USA, 99: 11890-11895, 2002) and
elsewhere. Further, a variety of commercially available kits for
constructing RNA are useful for making the RNA to be used in the
present invention. RNA is constructed from organs/tissues/cells
procured from normal healthy subjects; however, this invention
contemplates construction of RNA from diseased subjects. This
invention contemplates using any type of organ from any type of
subject or animal. For test samples RNA may be procured from an
individual (e.g., any animal, including mammals) with or without
visible disease and from tissue samples, biological fluids (e.g.,
whole blood) or the like. In some embodiments amplification or
construction of cDNA sequences may be helpful to increase detection
capabilities. The present invention, as well as the art, provides
the requisite level of detail to perform such tasks. In one aspect
of the present invention, whole blood is used as the source of RNA
and accordingly, RNA stabilizing regeants are optionally used, such
as PAX tubes, as described in Thach et al., J. Immunol. Methods.
December 283(1-2):269-279, 2003 and Chai et al., J. Clin. Lab Anal.
19(5):182-188, 2005 (both of which are incorporated herein by
reference in their entirety).
[0370] Complementary DNA (cDNA) libraries can be generated using
techniques known in the art, such as those described in Ausubel et
al. (2001 Current Protocols in Molecular Biology, Greene Publ.
Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y.); Sambrook
et al. (1989 Molecular Cloning, Second Ed., Cold Spring Harbor
Laboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular
Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.) and
elsewhere. Further, a variety of commercially available kits for
constructing cDNA libraries are useful for making the cDNA
libraries of the present invention. Libraries are constructed from
organs/tissues/cells procured from normal, healthy subjects.
Amplification or Nucleic Acid Amplification
[0371] By "amplification" or "nucleic acid amplification" is meant
production of multiple copies of a target nucleic acid that
contains at least a portion of the intended specific target nucleic
acid sequence. The multiple copies may be referred to as amplicons
or amplification products. In certain embodiments, the amplified
target contains less than the complete target gene sequence
(introns and exons) or an expressed target gene sequence (spliced
transcript of exons and flanking untranslated sequences). For
example, specific amplicons may be produced by amplifying a portion
of the target polynucleotide by using amplification primers that
hybridize to, and initiate polymerization from, internal positions
of the target polynucleotide. Preferably, the amplified portion
contains a detectable target sequence that may be detected using
any of a variety of well-known methods.
[0372] Many well-known methods of nucleic acid amplification
require thermocycling to alternately denature double-stranded
nucleic acids and hybridize primers; however, other well-known
methods of nucleic acid amplification are isothermal. The
polymerase chain reaction (U.S. Pat. Nos. 4,683,195; 4,683,202;
4,800,159; 4,965,188), commonly referred to as PCR, uses multiple
cycles of denaturation, annealing of primer pairs to opposite
strands, and primer extension to exponentially increase copy
numbers of the target sequence. In a variation called RT-PCR,
reverse transcriptase (RT) is used to make a complementary DNA
(cDNA) from mRNA, and the cDNA is then amplified by PCR to produce
multiple copies of DNA. The ligase chain reaction (Weiss, R. 1991,
Science 254: 1292), commonly referred to as LCR, uses two sets of
complementary DNA oligonucleotides that hybridize to adjacent
regions of the target nucleic acid. The DNA oligonucleotides are
covalently linked by a DNA ligase in repeated cycles of thermal
denaturation, hybridization and ligation to produce a detectable
double-stranded ligated oligonucleotide product. Another method is
strand displacement amplification (Walker, G. et al., 1992, Proc.
Natl. Acad. Sci. USA 89:392-396; U.S. Pat. Nos. 5,270,184 and
5,455,166), commonly referred to as SDA, which uses cycles of
annealing pairs of primer sequences to opposite strands of a target
sequence, primer extension in the presence of a dNTP.alpha.S to
produce a duplex hemiphosphorothioated primer extension product,
endonuclease-mediated nicking of a hemimodified restriction
endonuclease recognition site, and polymerase-mediated primer
extension from the 3' end of the nick to displace an existing
strand and produce a strand for the next round of primer annealing,
nicking and strand displacement, resulting in geometric
amplification of product. Thermophilic SDA (tSDA) uses thermophilic
endonucleases and polymerases at higher temperatures in essentially
the same method (European Pat. No. 0 684 315). Other amplification
methods include: nucleic acid sequence based amplification (U.S.
Pat. No. 5,130,238), commonly referred to as NASBA; one that uses
an RNA replicase to amplify the probe molecule itself (Lizardi, P.
et al., 1988, BioTechnol. 6: 1197-1202), commonly referred to as
Q.beta. replicase; a transcription based amplification method
(Kwoh, D. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177);
self-sustained sequence replication (Guatelli, J. et al., 1990,
Proc. Natl. Acad. Sci. USA 87: 1874-1878); and, transcription
mediated amplification (U.S. Pat. Nos. 5,480,784 and 5,399,491),
commonly referred to as TMA. For further discussion of known
amplification methods see Persing, David H., 1993, "In Vitro
Nucleic Acid Amplification Techniques" in Diagnostic Medical
Microbiology: Principles and Applications (Persing et al., Eds.),
pp. 51-87 (American Society for Microbiology, Washington,
D.C.).
[0373] Illustrative transcription-based amplification systems of
the present invention include TMA, which employs an RNA polymerase
to produce multiple RNA transcripts of a target region (U.S. Pat.
Nos. 5,480,784 and 5,399,491). TMA uses a "promoter-primer" that
hybridizes to a target nucleic acid in the presence of a reverse
transcriptase and an RNA polymerase to form a double-stranded
promoter from which the RNA polymerase produces RNA transcripts.
These transcripts can become templates for further rounds of TMA in
the presence of a second primer capable of hybridizing to the RNA
transcripts. Unlike PCR, LCR or other methods that require heat
denaturation, TMA is an isothermal method that uses an RNase H
activity to digest the RNA strand of an RNA:DNA hybrid, thereby
making the DNA strand available for hybridization with a primer or
promoter-primer. Generally, the RNase H activity associated with
the reverse transcriptase provided for amplification is used.
[0374] In an illustrative TMA method, one amplification primer is
an oligonucleotide promoter-primer that comprises a promoter
sequence which becomes functional when double-stranded, located 5'
of a target-binding sequence, which is capable of hybridizing to a
binding site of a target RNA at a location 3' to the sequence to be
amplified. A promoter-primer may be referred to as a "T7-primer"
when it is specific for T7 RNA polymerase recognition. Under
certain circumstances, the 3' end of a promoter-primer, or a
subpopulation of such promoter-primers, may be modified to block or
reduce primer extension. From an unmodified promoter-primer,
reverse transcriptase creates a cDNA copy of the target RNA, while
RNase H activity degrades the target RNA. A second amplification
primer then binds to the cDNA. This primer may be referred to as a
"non-T7 primer" to distinguish it from a "T7-primer". From this
second amplification primer, reverse transcriptase creates another
DNA strand, resulting in a double-stranded DNA with a functional
promoter at one end. When double-stranded, the promoter sequence is
capable of binding an RNA polymerase to begin transcription of the
target sequence to which the promoter-primer is hybridized. An RNA
polymerase uses this promoter sequence to produce multiple RNA
transcripts (i.e., amplicons), generally about 100 to 1,000 copies.
Each newly-synthesized amplicon can anneal with the second
amplification primer. Reverse transcriptase can then create a DNA
copy, while the RNase H activity degrades the RNA of this RNA:DNA
duplex. The promoter-primer can then bind to the newly synthesized
DNA, allowing the reverse transcriptase to create a double-stranded
DNA, from which the RNA polymerase produces multiple amplicons.
Thus, a billion-fold isothermic amplification can be achieved using
two amplification primers.
[0375] "Selective amplification", as used herein, refers to the
amplification of a target nucleic acid sequence according to the
present invention wherein detectable amplification of the target
sequence is substantially limited to amplification of target
sequence contributed by a nucleic acid sample of interest that is
being tested and is not contributed by target nucleic acid sequence
contributed by some other sample source, e.g., contamination
present in reagents used during amplification reactions or in the
environment in which amplification reactions are performed.
[0376] By "amplification conditions" is meant conditions permitting
nucleic acid amplification according to the present invention.
Amplification conditions may, in some embodiments, be less
stringent than "stringent hybridization conditions" as described
herein. Oligonucleotides used in the amplification reactions of the
present invention hybridize to their intended targets under
amplification conditions, but may or may not hybridize under
stringent hybridization conditions. On the other hand, detection
probes of the present invention hybridize under stringent
hybridization conditions. While the Examples section infra provides
preferred amplification conditions for amplifying target nucleic
acid sequences according to the present invention, other acceptable
conditions to carry out nucleic acid amplifications according to
the present invention could be easily ascertained by someone having
ordinary skill in the art depending on the particular method of
amplification employed.
Oligonucleotides & Primers for Amplification
[0377] As used herein, the term "oligonucleotide" or "oligo" or
"oligomer" is intended to encompass a singular "oligonucleotide" as
well as plural "oligonucleotides," and refers to any polymer of two
or more of nucleotides, nucleosides, nucleobases or related
compounds used as a reagent in the amplification methods of the
present invention, as well as subsequent detection methods. The
oligonucleotide may be DNA and/or RNA and/or analogs thereof. The
term oligonucleotide does not denote any particular function to the
reagent, rather, it is used generically to cover all such reagents
described herein. An oligonucleotide may serve various different
functions, e.g., it may function as a primer if it is capable of
hybridizing to a complementary strand and can further be extended
in the presence of a nucleic acid polymerase, it may provide a
promoter if it contains a sequence recognized by an RNA polymerase
and allows for transcription, and it may function to prevent
hybridization or impede primer extension if appropriately situated
and/or modified. Specific oligonucleotides of the present invention
are described in more detail below, but are directed to binding the
organ-specific transcript or the organ-specific transcript encoding
the sequences listed in the attached Tables 1-32, 36-45 and 47-79
or the appended sequence listing. As used herein, an
oligonucleotide can be virtually any length, limited only by its
specific function in the amplification reaction or in detecting an
amplification product of the amplification reaction.
[0378] Oligonucleotides of a defined sequence and chemical
structure may be produced by techniques known to those of ordinary
skill in the art, such as by chemical or biochemical synthesis, and
by in vitro or in vivo expression from recombinant nucleic acid
molecules, e.g., bacterial or viral vectors. As intended by this
disclosure, an oligonucleotide does not consist solely of wild-type
chromosomal DNA or the in vivo transcription products thereof.
[0379] Oligonucleotides may be modified in any way, as long as a
given modification is compatible with the desired function of a
given oligonucleotide. One of ordinary skill in the art can easily
determine whether a given modification is suitable or desired for
any given oligonucleotide of the present invention. Modifications
include base modifications, sugar modifications or backbone
modifications. Base modifications include, but are not limited to
the use of the following bases in addition to adenine, cytidine,
guanosine, thymine and uracil: C-5 propyne, 2-amino adenine,
5-methyl cytidine, inosine, and dP and dK bases. The sugar groups
of the nucleoside subunits may be ribose, deoxyribose and analogs
thereof, including, for example, ribonucleosides having a
2'-O-methyl substitution to the ribofuranosyl moiety. See Becker et
al., U.S. Pat. No. 6,130,038. Other sugar modifications include,
but are not limited to 2'-amino, 2'-fluoro,
(L)-alpha-threofuranosyl, and pentopuranosyl modifications. The
nucleoside subunits may by joined by linkages such as
phosphodiester linkages, modified linkages or by non-nucleotide
moieties which do not prevent hybridization of the oligonucleotide
to its complementary target nucleic acid sequence. Modified
linkages include those linkages in which a standard phosphodiester
linkage is replaced with a different linkage, such as a
phosphorothioate linkage or a methylphosphonate linkage. The
nucleobase subunits may be joined, for example, by replacing the
natural deoxyribose phosphate backbone of DNA with a pseudo peptide
backbone, such as a 2-aminoethylglycine backbone which couples the
nucleobase subunits by means of a carboxymethyl linker to the
central secondary amine. (DNA analogs having a pseudo peptide
backbone are commonly referred to as "peptide nucleic acids" or
"PNA" and are disclosed by Nielsen et al., "Peptide Nucleic Acids,"
U.S. Pat. No. 5,539,082.) Other linkage modifications include, but
are not limited to, morpholino bonds.
[0380] Non-limiting examples of oligonucleotides or oligomers
contemplated by the present invention include nucleic acid analogs
containing bicyclic and tricyclic nucleoside and nucleotide analogs
(LNAs). See Imanishi et al., U.S. Pat. No. 6,268,490; and Wengel et
al., U.S. Pat. No. 6,670,461.) Any nucleic acid analog is
contemplated by the present invention provided the modified
oligonucleotide can perform its intended function, e.g., hybridize
to a target nucleic acid under stringent hybridization conditions
or amplification conditions, or interact with a DNA or RNA
polymerase, thereby initiating extension or transcription. In the
case of detection probes, the modified oligonucleotides must also
be capable of preferentially hybridizing to the target nucleic acid
under stringent hybridization conditions.
[0381] While design and sequence of oligonucleotides for the
present invention depend on their function as described below,
several variables must generally be taken into account. Among the
most critical are: length, melting temperature (Tm), specificity,
complementarity with other oligonucleotides in the system, G/C
content, polypyrimidine (T, C) or polypurine (A, G) stretches, and
the 3'-end sequence. Controlling for these and other variables is a
standard and well known aspect of oligonucleotide design, and
various computer programs are readily available to screen large
numbers of potential oligonucleotides for optimal ones.
[0382] The 3'-terminus of an oligonucleotide (or other nucleic
acid) can be blocked in a variety of ways using a blocking moiety,
as described below. A "blocked" oligonucleotide is not efficiently
extended by the addition of nucleotides to its 3'-terminus, by a
DNA- or RNA-dependent DNA polymerase, to produce a complementary
strand of DNA. As such, a "blocked" oligonucleotide cannot be a
"primer."
[0383] As used in this disclosure, the phrase "an oligonucleotide
having a nucleic acid sequence `comprising,` `consisting of,` or
`consisting essentially of` a sequence selected from" a group of
specific sequences means that the oligonucleotide, as a basic and
novel characteristic, is capable of stably hybridizing to a nucleic
acid having the exact complement of one of the listed nucleic acid
sequences of the group under stringent hybridization conditions. An
exact complement includes the corresponding DNA or RNA
sequence.
[0384] The phrase "an oligonucleotide substantially corresponding
to a nucleic acid sequence" means that the referred to
oligonucleotide is sufficiently similar to the reference nucleic
acid sequence such that the oligonucleotide has similar
hybridization properties to the reference nucleic acid sequence in
that it would hybridize with the same target nucleic acid sequence
under stringent hybridization conditions.
[0385] One skilled in the art will understand that "substantially
corresponding" oligonucleotides of the invention can vary from the
referred to sequence and still hybridize to the same target nucleic
acid sequence. This variation from the nucleic acid may be stated
in terms of a percentage of identical bases within the sequence or
the percentage of perfectly complementary bases between the probe
or primer and its target sequence. Thus, an oligonucleotide of the
present invention substantially corresponds to a reference nucleic
acid sequence if these percentages of base identity or
complementarity are from 100% to about 80%. In preferred
embodiments, the percentage is from 100% to about 85%. In more
preferred embodiments, this percentage can be from 100% to about
90%; in other preferred embodiments, this percentage is from 100%
to about 95%. One skilled in the art will understand the various
modifications to the hybridization conditions that might be
required at various percentages of complementarity to allow
hybridization to a specific target sequence without causing an
unacceptable level of non-specific hybridization.
[0386] The skilled artisan will recognize that any of a wide
variety of known and available amplification techniques may be
employed in the methods of the present invention, even if not
explicitly described herein. Illustrative non-limiting examples of
such amplification techniques are described below.
[0387] One illustrative amplification technique useful in
accordance with the methods herein is the polymerase chain
reaction. As noted above, the polymerase chain reaction (U.S. Pat.
Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188), commonly referred
to as PCR, uses multiple cycles of denaturation, annealing of
primer pairs to opposite strands, and primer extension to
exponentially increase copy numbers of the target sequence. In a
variation called RT-PCR, reverse transcriptase (RT) is used to make
a complementary DNA (cDNA) from mRNA, and the cDNA is then
amplified by PCR to produce multiple copies of DNA.
[0388] Another illustrative amplification method, the ligase chain
reaction (Weiss, R. 1991, Science 254: 1292), commonly referred to
as LCR, uses two sets of complementary DNA oligonucleotides that
hybridize to adjacent regions of the target nucleic acid. The DNA
oligonucleotides are covalently linked by a DNA ligase in repeated
cycles of thermal denaturation, hybridization and ligation to
produce a detectable double-stranded ligated oligonucleotide
product.
[0389] Another illustrative method is strand displacement
amplification (Walker, G. et al., 1992, Proc. Natl. Acad. Sci. USA
89:392-396; U.S. Pat. Nos. 5,270,184 and 5,455,166), commonly
referred to as SDA, which uses cycles of annealing pairs of primer
sequences to opposite strands of a target sequence, primer
extension in the presence of a dNTP S to produce a duplex
hemiphosphorothioated primer extension product,
endonuclease-mediated nicking of a hemimodified restriction
endonuclease recognition site, and polymerase-mediated primer
extension from the 3' end of the nick to displace an existing
strand and produce a strand for the next round of primer annealing,
nicking and strand displacement, resulting in geometric
amplification of product. Thermophilic SDA (tSDA) uses thermophilic
endonucleases and polymerases at higher temperatures in essentially
the same method (European Pat. No. 0 684 315).
[0390] Other amplification methods include, for example, nucleic
acid sequence based amplification (U.S. Pat. No. 5,130,238),
commonly referred to as NASBA; one that uses an RNA replicase to
amplify the probe molecule itself (Lizardi, P. et al., 1988,
BioTechnol. 6: 1197-1202), commonly referred to as Q replicase; a
transcription based amplification method (Kwoh, D. et al., 1989,
Proc. Natl. Acad. Sci. USA 86:1173-1177); self-sustained sequence
replication (Guatelli, J. et al., 1990, Proc. Natl. Acad. Sci. USA
87: 1874-1878); and, transcription mediated amplification (U.S.
Pat. Nos. 5,480,784 and 5,399,491), commonly referred to as TMA.
For further discussion of known amplification methods see Persing,
David H., 1993, "In Vitro Nucleic Acid Amplification Techniques" in
Diagnostic Medical Microbiology: Principles and Applications
(Persing et al., Eds.), pp. 51-87 (American Society for
Microbiology, Washington, D.C.).
[0391] In more particular embodiments, the amplification technique
used in the methods of the present invention is a
transcription-based amplification technique, such as TMA and
NASBA.
[0392] All or substantially all of the unique transcripts of RNA or
from a cDNA library, e.g., representing virtually or substantially
all genes functioning in the organ of interest, can be identified
and quantified using any of a variety of techniques known in the
art. In this regard, in certain embodiments, substantially all
refers to a sample representing at least 80% of all genes
detectably expressed in the organ of interest. In a further
embodiment, substantially all refers to a sample representing at
least 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher of
all genes functioning in the organ of interest. In one embodiment,
substantially all the transcripts from a cDNA library are
amplified, sorted and signature sequences generated therefrom
according to the methods described in U.S. Pat. Nos. 6,013,445;
6,172,218; 6,172,214; 6,140,489 and Brenner, P., et al., Nat
Biotechnol, 18:630-634 2000. Briefly, polynucleotide templates from
a cDNA library of interest are cloned into a vector system that
contains a vast set of minimally cross-hybridizing oligonucleotide
tags (see U.S. Pat. No. 5,863,722). The number of tags is usually
at least 100 times greater than the number of cDNA templates (see
e.g., U.S. Pat. No. 6,013,445 and Brenner, P., et al., supra).
Thus, the set of tags is such that a 1% sample taken of
template-tag conjugates ensures that essentially every template in
the sample is conjugated to a unique tag and that at least one of
each of the different template cDNAs is represented in the sample
with >99% probability (U.S. Pat. No. 6,013,445 and Brenner, P.,
et al., supra). The conjugates are then amplified and hybridized
under stringent conditions to microbeads each of which has attached
thereto a unique complementary, minimally cross-hybridizing
oligonucleotide tag. The transcripts are then directly sequenced
simultaneously in a flow cell using a ligation-based sequencing
method (see e.g., U.S. Pat. No. 6,013,445). A short signature
sequence of about 16-20 base pairs (Brenner, P., et al., supra) is
generated simultaneously from each of the hundreds of thousands of
beads (or more) in the flow cell, each having attached thereto
copies of a unique transcript from the sample. This technique is
termed massively parallel signature sequencing (MPSS).
[0393] The resulting sequences, (e.g., MPSS signature sequences),
are generally about 17-20 bases in length. However, in certain
embodiments, the sequences can be about 8, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more
bases in length. The sequences are annotated using annotated human
genome sequence (such as human genome release hg16, released in
November, 2003, or other public or private databases) and the human
Unigene (Unigene build #184) using methods known in the art, such
as the method described by Meyers, B. C., et al., Genome Res, 14:
1641-1653, 2004. Other databases useful in this regard include
Genbank, EMBL, or other publicly available databases. In certain
embodiments, transcripts are considered only for those with 100%
matches between an MPSS or other type of signature and a genome
signature. As would be readily appreciated by the skilled artisan
upon reading the present disclosure, this is a stringent match
criterion and in certain embodiments, it may be desirable to use
less stringent match criteria. Indeed, polymorphisms could lead to
variations in transcripts that would be missed if only exact
matches were used. For example, it may be desirable to consider
signature sequences that match a genome signature with 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment,
signatures that are expressed at less than 3 transcripts per
million in libraries of interest are disregarded, as they might not
be reliably detected since this, in effect, represents less than
one transcript per cell (see for example, Jongeneel, C. V., et al.,
Proc Natl Acad Sci USA, 2003). Alternatively, transcripts at this
level may arise from cells that are present as only a fraction of
the population (e.g., 1%)--hence the measurement could be real.
cDNA signatures are classified by their positions relative to
polyadenylation signals and poly (A) tails and by their orientation
relative to the 5*3 orientation of source mRNA. Full-length
sequences corresponding to the signature sequences can be thus
identified.
[0394] In one embodiment, substantially all the transcripts from a
cDNA library are identified using sequencing by synthesis (SBS) or
similar technology, such as that developed by Solexa (now part of
Illumina) (San Diego, Calif.). This technology may be used to
identify signature sequences of the transcriptome of a particular
organ/tissue/cell of interest. See for example, the methods
described in Expert Rev Mol. Diagn. 2007 January; 7(1):65-76;
Rosenthal, A & Brenner, S. 1994-2000. U.S. Pat. No. 6,087,095
DNA sequencing method; Ronaghi, M., Uhlen, M., and Nyren, P. 1998.
Science 281: 363. A sequencing method based on real-time
pyrophosphate.; Mitra, R D, Shendure, J, Olejnik, J, Olejnik, E K,
and Church, G M 2003 Analyt. Biochem. 320:55-65 Fluorescent in situ
Sequencing on Polymerase Colonies; Johnson D S, Mortazavi A, Myers
R M, Wold B. (2007) Genome-wide mapping of in vivo protein-DNA
interactions. Science 316(5830):1441-2; A. Barski et al., 2007 Cell
129, 823-837; T. Mikkelsen et al., Nature. 2007 448(7153):553-60;
G. Robertson et al., Nature Methods 2007 August; 4(8):651-7; R.F.
Service 2006 Science 311, 1544-1546; and U.S. Pat. Nos. 7,232,656;
7,115,400; 7,057,026; 6,969,488; 6,897,023; 6,833,246.
[0395] In certain embodiments, other techniques may be used to
evaluate RNA transcripts of the transcripts from a particular cDNA
library, including microarray analysis (Han, M., et al., Nat
Biotechnol, 19: 631-635, 2001; Bao, P., et al., Anal Chem, 74:
1792-1797, 2002; Schena et al., Proc. Natl. Acad. Sci. USA
93:10614-19, 1996; and Heller et al., Proc. Natl. Acad. Sci. USA
94:2150-55, 1997) and SAGE (serial analysis of gene expression).
Like MPSS, SAGE is digital and can generate a large number of
signature sequences. (see e.g., Velculescu, V. E., et al., Trends
Genet, 16: 423-425, 2000; Tuteja R. and Tuteja N. Bioessays. 2004
August; 26(8):916-22), although orders of magnitude fewer than that
are available from techniques such as MPSS.
[0396] As one of skill in the art could readily appreciate any
number of methodologies can be employed to investigate the
organ-specific nucleic acid and polypeptide sequences set forth by
the present invention. In addition to protein or nucleic acid array
or microarray analysis, other nanoscale analysis may be employed.
Such methodologies include, but are not limited to microfluidic
platforms, nanowire sensors (Bunimovich et al., Electrochemically
Programmed, Spatially Selective Biofunctionalization of Silicon
Wires, Langmuir 20, 10630-10638, 2004; Curreli et al., J. Am. Chem.
Soc. 127, 6922-6923, 2005). Further, the use of high-affinity
protein-capture agents is contemplated. Such capture agents may
include DNA aptamers (U.S. Patent Application Pub. No. 20030219801,
as well as the use of click chemistry for target-guided synthesis
(Lewis et al., Angewandte Chemie-International Edition, 41, 1053-,
2002; Manetsch et al., J. Am. Chem. Soc. 126, 12809-12818, 2004;
Ramstrom et al., Nature Rev. Drug Discov. 1, 26-36, 2002).
[0397] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques and descriptions of
organic chemistry, polymer technology, molecular biology (including
recombinant techniques), cell biology, biochemistry, and
immunology, which are within the skill of the art. Such
conventional techniques include polymer array synthesis,
hybridization, ligation, and detection of hybridization using a
label. Specific illustrations of suitable techniques can be had by
reference to the example herein below. However, other equivalent
conventional procedures can, of course, also be used. Such
conventional techniques and descriptions can be found in standard
laboratory manuals such as Genome Analysis: A Laboratory Manual
Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells:
A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular
Cloning: A Laboratory Manual (all from Cold Spring Harbor
Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.)
Freeman, New York, Gait, "Oligonucleotide Synthesis: A Practical
Approach" 1984, IRL Press, London, Nelson and Cox (2000),
Lehninger, Principles of Biochemistry .sup.3rd Ed., W.H. Freeman
Pub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5th Ed.,
W.H. Freeman Pub., New York, N.Y., all of which are herein
incorporated in their entirety by reference for all purposes.
[0398] The present invention can employ solid substrates, including
arrays in some preferred embodiments. Methods and techniques
applicable to polymer (including protein) array synthesis have been
described in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos.
5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783,
5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215,
5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734,
5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324,
5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860,
6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT
Applications Nos. PCT/US99/00730 (International Publication No. WO
99/36760) and PCT/US01/04285 (International Publication No. WO
01/58593), which are all incorporated herein by reference in their
entirety for all purposes. Patents that describe synthesis
techniques in specific embodiments include U.S. Pat. Nos.
5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and
5,959,098.
[0399] Nucleic acid arrays that are useful in the present invention
include those known in the art and that can be manufactured using
the cognate sequences to those organ-specific nucleic acid
sequences and nucleic acid encoding sequence set forth in Tables
1-32, 36-45 and 47-79 and the attached sequence listing, as well as
those that are commercially available from Affymetrix (Santa Clara,
Calif.) under the brand name GeneChip.TM.. Example arrays are shown
on the website at affymetrix.com. Further exemplary methods of
manufacturing and using arrays are provided in, for example, U.S.
Pat. Nos. 7,028,629; 7,011,949; 7,011,945; 6,936,419; 6,927,032;
6,924,103; 6,921,642; and 6,818,394 to name a few.
[0400] The present invention as related to arrays and microarrays
also contemplates many uses for polymers attached to solid
substrates. These uses include gene expression monitoring,
profiling, library screening, genotyping and diagnostics. Gene
expression monitoring and profiling methods and methods useful for
gene expression monitoring and profiling are shown in U.S. Pat.
Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138,
6,177,248 and 6,309,822. Genotyping and uses therefore are shown in
U.S. Ser. Nos. 10/442,021, 10/013,598 (U.S. Patent Application
Publication 20030036069), and U.S. Pat. Nos. 5,925,525, 6,268,141,
5,856,092, 6,267,152, 6,300,063, 6,525,185, 6,632,611, 5,858,659,
6,284,460, 6,361,947, 6,368,799, 6,673,579 and 6,333,179. Other
methods of nucleic acid amplification, labeling and analysis that
may be used in combination with the methods disclosed herein are
embodied in U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996,
5,541,061, and 6,197,506.
[0401] The present invention also contemplates sample preparation
methods in certain preferred embodiments. Prior to or concurrent
with analysis, the genomic sample may be amplified by a variety of
mechanisms, some of which may employ PCR. See, for example, PCR
Technology: Principles and Applications for DNA Amplification (Ed.
H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A
Guide to Methods and Applications (Eds. Innis, et al., Academic
Press, San Diego, Calif., 1990); Mattila et al., NucleicAcids Res.
19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17
(1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S.
Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188, and 5,333,675,
and each of which is incorporated herein by reference in their
entireties for all purposes. Modifications to PCR may also be used,
for example, the inclusion of Betaine or trimethylglycine, which
has been disclosed, for example, in Rees et al. Biochemistry
32:137-144 (1993), and in U.S. Pat. Nos. 6,270,962 and 5,545,539.
The sample may be amplified on the array. See, for example, U.S.
Pat. No. 6,300,070 and U.S. Ser. No. 09/513,300, which are
incorporated herein by reference.
[0402] Other suitable amplification methods include the ligase
chain reaction (LCR) (for example, Wu and Wallace, Genomics 4, 560
(1989), Landegren et al., Science 241, 1077 (1988) and Barringer et
al. Gene 89:117 (1990)), transcription amplification (Kwoh et al.,
Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315),
self-sustained sequence replication (Guatelli et al., Proc. Nat.
Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective
amplification of target polynucleotide sequences (U.S. Pat. No.
6,410,276), consensus sequence primed polymerase chain reaction
(CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase
chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)
nucleic acid based sequence amplification (NABSA), rolling circle
amplification (RCA), multiple displacement amplification (MDA)
(U.S. Pat. Nos. 6,124,120 and 6,323,009) and circle-to-circle
amplification (C2CA) (Dahl et al. Proc. Natl. Acad. Sci.
101:4548-4553 (2004). (See, U.S. Pat. Nos. 5,409,818, 5,554,517,
and 6,063,603, each of which is incorporated herein by reference).
Other amplification methods that may be used are described in, U.S.
Pat. Nos. 5,242,794, 5,494,810, 5,409,818, 4,988,617, 6,063,603 and
5,554,517 and in U.S. Ser. No. 09/854,317, each of which is
incorporated herein by reference.
[0403] Additional methods of sample preparation and techniques for
reducing the complexity of a nucleic sample are described in Dong
et al., Genome Research 11, 1418 (2001), in U.S. Pat. Nos.
6,361,947, 6,391,592 and U.S. Ser. Nos. 09/916,135, 09/920,491
(U.S. Patent Application Publication 20030096235), Ser. No.
09/910,292 (U.S. Patent Application Publication 20030082543), and
Ser. No. 10/013,598.
[0404] Methods for conducting polynucleotide hybridization assays
have been well developed in the art. Hybridization assay procedures
and conditions will vary depending on the application and are
selected in accordance with the general binding methods known
including those referred to in: Maniatis et al. Molecular Cloning:
A Laboratory Manual (2nd Ed. Cold Spring Harbor, N.Y., 1989);
Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to
Molecular Cloning Techniques (Academic Press, Inc., San Diego,
Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983). Methods
and apparatus for carrying out repeated and controlled
hybridization reactions have been described in U.S. Pat. Nos.
5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of
which are incorporated herein by reference
[0405] The present invention also contemplates signal detection of
hybridization between ligands in certain preferred embodiments. See
U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758;
5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639;
6,218,803; and 6,225,625, in U.S. Ser. No. 10/389,194 and in PCT
Application PCT/US99/06097 (published as WO99/47964), each of which
also is hereby incorporated by reference in its entirety for all
purposes.
[0406] Methods and apparatus for signal detection and processing of
intensity data are disclosed in, for example, U.S. Pat. Nos.
5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758;
5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555,
6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S.
Ser. Nos. 10/389,194, 60/493,495 and in PCT Application
PCT/US99/06097 (published as WO99/47964), each of which also is
hereby incorporated by reference in its entirety for all
purposes.
[0407] The practice of the present invention may also employ
conventional biology methods, software and systems. Computer
software products of the invention typically include computer
readable medium having computer-executable instructions for
performing the logic steps of the method of the invention. Suitable
computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM,
hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The
computer executable instructions may be written in a suitable
computer language or combination of several languages. Basic
computational biology methods are described in, for example Setubal
and Meidanis et al., Introduction to Computational Biology Methods
(PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif,
(Ed.), Computational Methods in Molecular Biology, (Elsevier,
Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:
Application in Biological Science and Medicine (CRC Press, London,
2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide
for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2nd ed.,
2001). See U.S. Pat. No. 6,420,108.
[0408] The present invention may also make use of various computer
program products and software for a variety of purposes, such as
probe design, management of data, analysis, and instrument
operation. See, U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729,
5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127,
6,229,911 and 6,308,170.
[0409] The whole genome sampling assay (WGSA) is described, for
example in Kennedy et al., Nat. Biotech. 21, 1233-1237 (2003),
Matsuzaki et al., Gen. Res. 14: 414-425, (2004), and Matsuzaki, et
al. Nature Methods 1:109-111 (2004). Algorithms for use with
mapping assays are described, for example, in Liu et al.,
Bioinformatics 19: 2397-2403 (2003) and Di et al. Bioinformatics
21:1958 (2005). Additional methods related to WGSA and arrays
useful for WGSA and applications of WGSA are disclosed, for
example, in U.S. Patent Application Nos. 60/676,058 filed Apr. 29,
2005, 60/616,273 filed Oct. 5, 2004, Ser. Nos. 10/912,445,
11/044,831, 10/442,021, 10/650,332 and 10/463,991. Genome wide
association studies using mapping assays are described in, for
example, Hu et al., Cancer Res.; 65(7):2542-6 (2005), Mitra et al.,
Cancer Res., 64(21):8116-25 (2004), Butcher et al., Hum Mol.
Genet., 14(10):1315-25 (2005), and Klein et al., Science,
308(5720):385-9 (2005). Each of these references is incorporated
herein by reference in its entirety for all purposes.
[0410] Additionally, the present invention may have preferred
embodiments that include methods for providing genetic information
over networks such as the Internet as shown in U.S. Ser. Nos.
10/197,621, 10/063,559 (United States Publication Number
20020183936), Ser. Nos. 10/065,856, 10/065,868, 10/328,818,
10/328,872, 10/423,403, and 60/482,389.
[0411] The term "array" as used herein refers to an intentionally
created collection of molecules that can be prepared either
synthetically or biosynthetically. The molecules in the array can
be identical or different from each other. The array can assume a
variety of formats, for example, libraries of soluble molecules;
libraries of compounds tethered to resin beads, silica chips, or
other solid supports.
[0412] The term "mRNA" or sometimes refer by "mRNA transcripts" as
used herein, include, but not limited to pre-mRNA transcript(s),
transcript processing intermediates, mature mRNA(s) ready for
translation and transcripts of the gene or genes, or nucleic acids
derived from the mRNA transcript(s). Transcript processing may
include splicing, editing and degradation. As used herein, a
nucleic acid derived from an mRNA transcript refers to a nucleic
acid for whose synthesis the mRNA transcript or a subsequence
thereof has ultimately served as a template. Thus, a cDNA reverse
transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA
amplified from the cDNA, an RNA transcribed from the amplified DNA,
etc., are all derived from the mRNA transcript and detection of
such derived products is indicative of the presence and/or
abundance of the original transcript in a sample. Thus, mRNA
derived samples include, but are not limited to, mRNA transcripts
of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA
transcribed from the cDNA, DNA amplified from the genes, RNA
transcribed from amplified DNA, and the like.
[0413] The term "nucleic acid library" or sometimes refer by
"array" as used herein refers to an intentionally created
collection of nucleic acids which can be prepared either
synthetically or biosynthetically and screened for biological
activity in a variety of different formats (for example, libraries
of soluble molecules; and libraries of oligos tethered to resin
beads, silica chips, or other solid supports). Additionally, the
term "array" is meant to include those libraries of nucleic acids
which can be prepared by spotting nucleic acids of essentially any
length (for example, from 1 to about 1000 nucleotide monomers in
length) onto a substrate. The term "nucleic acid" as used herein
refers to a polymeric form of nucleotides of any length, either
ribonucleotides, deoxyribonucleotides or peptide nucleic acids
(PNAs), that comprise purine and pyrimidine bases, or other
natural, chemically or biochemically modified, non-natural, or
derivatized nucleotide bases. The backbone of the polynucleotide
can comprise sugars and phosphate groups, as may typically be found
in RNA or DNA, or modified or substituted sugar or phosphate
groups. A polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. The sequence of
nucleotides may be interrupted by non-nucleotide components. Thus
the terms nucleoside, nucleotide, deoxynucleoside and
deoxynucleotide generally include analogs such as those described
herein. These analogs are those molecules having some structural
features in common with a naturally occurring nucleoside or
nucleotide such that when incorporated into a nucleic acid or
oligonucleoside sequence, they allow hybridization with a naturally
occurring nucleic acid sequence in solution. Typically, these
analogs are derived from naturally occurring nucleosides and
nucleotides by replacing and/or modifying the base, the ribose or
the phosphodiester moiety. The changes can be tailor made to
stabilize or destabilize hybrid formation or enhance the
specificity of hybridization with a complementary nucleic acid
sequence as desired.
[0414] The term "nucleic acids" as used herein may include any
polymer or oligomer of pyrimidine and purine bases, preferably
cytosine, thymine, and uracil, and adenine and guanine,
respectively. See Albert L. Lehninger, PRINCIPLES OF BIOCHEMISTRY,
at 793-800 (Worth Pub. 1982). Indeed, the present invention
contemplates any deoxyribonucleotide, ribonucleotide or peptide
nucleic acid component, and any chemical variants thereof, such as
methylated, hydroxymethylated or glucosylated forms of these bases,
and the like. The polymers or oligomers may be heterogeneous or
homogeneous in composition, and may be isolated from
naturally-occurring sources or may be artificially or synthetically
produced. In addition, the nucleic acids may be DNA or RNA, or a
mixture thereof, and may exist permanently or transitionally in
single-stranded or double-stranded form, including homoduplex,
heteroduplex, and hybrid states.
[0415] When referring to arrays and microarrays the term
"oligonucleotide" or sometimes refer by "polynucleotide" as used
herein refers to a nucleic acid ranging from at least 2, preferable
at least 8, and more preferably at least 20 nucleotides in length
or a compound that specifically hybridizes to a polynucleotide.
Polynucleotides of the present invention include sequences of
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may be
isolated from natural sources, recombinantly produced or
artificially synthesized and mimetics thereof. A further example of
a polynucleotide of the present invention may be peptide nucleic
acid (PNA). The invention also encompasses situations in which
there is a nontraditional base pairing such as Hoogsteen base
pairing which has been identified in certain tRNA molecules and
postulated to exist in a triple helix. "Polynucleotide" and
"oligonucleotide" are used interchangeably in this application.
[0416] The term "primer" as used herein refers to a single-stranded
oligonucleotide capable of acting as a point of initiation for
template-directed DNA synthesis under suitable conditions for
example, buffer and temperature, in the presence of four different
nucleoside triphosphates and an agent for polymerization, such as,
for example, DNA or RNA polymerase or reverse transcriptase. The
length of the primer, in any given case, depends on, for example,
the intended use of the primer, and generally ranges from 15 to 30
nucleotides. Short primer molecules generally require cooler
temperatures to form sufficiently stable hybrid complexes with the
template. A primer need not reflect the exact sequence of the
template but must be sufficiently complementary to hybridize with
such template. The primer site is the area of the template to which
a primer hybridizes. The primer pair is a set of primers including
a 5' upstream primer that hybridizes with the 5' end of the
sequence to be amplified and a 3' downstream primer that hybridizes
with the complement of the 3' end of the sequence to be
amplified.
[0417] The term "probe" as used herein refers to a
surface-immobilized molecule that can be recognized by a particular
target. See U.S. Pat. No. 6,582,908 for an example of arrays having
all possible combinations of probes with 10, 12, and more bases.
Examples of probes that can be investigated by this invention
include, but are not restricted to, agonists and antagonists for
cell membrane receptors, toxins and venoms, viral epitopes,
hormones (for example, opioid peptides, steroids, etc.), hormone
receptors, peptides, enzymes, enzyme substrates, cofactors, drugs,
lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides,
proteins, and monoclonal antibodies.
[0418] Also contemplated by the present invention are
polypeptide/protein arrays and microarrays. In certain embodiments,
such arrays comprise probes such as antibodies, aptamers, other
cognate binding ligands and the like specific to a component of the
sets disclosed herein. For example, such probes are specific to the
nucleic acid or polypeptide sequence set forth in Tables 1-32,
36-45 and 47-79 or the attached sequence listing. Such arrays and
methods of constructing the same are well known in the art, for
example, U.S. Pat. Publ. Nos. 20060035277; 20060166227;
20050260653; 20040199945; 20030044320; 20020102605; and U.S. Pat.
Nos. 6,777,239; 6,696,620; 6,689,568; 6,448,387; and 5,081,584.
[0419] One class of protein microarray useful in the context of the
present invention uses an immobilized "capture antibody." The
polypeptides are bound to a solid substrate, such as glass with a
treated surface, such as aminosilane or via a biotin-streptavidin
conjugation. The arrays are then incubated with a
solution-containing probe that will bind to the capture antibodies
in a manner dependent upon time, buffer components, and recognition
specificity. The probes may then be visualized directly if they
have been previously labeled, or may be allowed to bind to a
secondary labeled reagent, frequently another antibody. The means
of visualizing the amount of probe bound to the capture antibody is
dependent upon the labeling method utilized, but is often by a CCD
imager or laser scanner using filter sets that are appropriate to
excite and detect the emissions of the label. The imager converts
the amount of detected photons into an electronic signal (often an
8-bit or 16-bit scale) which can then be analyzed using software
packages.
[0420] In another embodiment, the present invention also provides a
protein-coated substrate comprising a plurality of patches arranged
in discrete, known regions on a substrate, where each of the
patches comprises an immobilized protein with a different, known
sequence and where each of the patches is separated from the
neighboring patches by from about 50 nm to about 500 .mu.m. In a
preferred embodiment, the protein-coated substrate comprises 9 or
more patches.
[0421] Arrays of proteins are also provided by the present
invention. In one embodiment, the protein arrays comprise
micrometer-scale, two-dimensional patterns of proteins immobilized
on arrays of functionalized surface patches.
[0422] In one embodiment, the array of proteins comprises a
plurality of patches, preferably 9 or more, arranged in discrete
known regions on a substrate, wherein each of the patches comprises
an immobilized protein with a different, known sequence and wherein
each of the patches is separated from neighboring patches by from
about 50 nm to about 500 .mu.m. In a preferred embodiment, the
patches are separated from neighboring patches from about 200 nm to
about 500 .mu.m.
[0423] In some versions of the array, the diameter of each of the
patches is proportional to the distance separating the patches.
Therefore, the area of each patch may be from about 100 nm.sup.2 to
about 40,000 .mu.m.sup.2. Each patch preferably has an area from
about 1 .mu.m.sup.2 to about 10,000 .mu.m.sup.2.
[0424] In one embodiment of the array, the array comprises 9 or
more patches within a total area of 1 cm.sup.2. In preferred
embodiments of the array, the array comprises 100 or more patches
within a total area of 1 cm.sup.2. In another embodiment, the array
comprises or more patches within a total area of 1 cm.sup.2.
[0425] In one embodiment of the array, the protein immobilized on
one patch differs from the protein immobilized on a second patch of
the same array.
[0426] In an alternative embodiment of the invention array, the
proteins on different patches are identical.
[0427] The substrate of the array may be either organic or
inorganic, biological or non-biological or any combination of these
materials. In one embodiment, the substrate is transparent or
translucent. The portion of the surface of the substrate on which
the patches reside is preferably flat and firm or semi-firm.
Numerous materials are suitable for use as a substrate in the array
embodiment of the invention. For instance, the substrate of the
invention array can comprise a material selected from a group
consisting of silicon, silica, quartz, glass, controlled pore
glass, carbon, alumina, titanium dioxide, germanium, silicon
nitride, zeolites, and gallium arsenide. Many metals such as gold,
platinum, aluminum, copper, titanium, and their alloys are also
options for substrates of the array. In addition, many ceramics and
polymers may also be used as substrates. Polymers which may be used
as substrates include, but are not limited to, the following:
polystyrene; poly(tetra)fluorethylene; (poly)vinylidenedifluoride;
polycarbonate; polymethylmethacrylate; polyvinylethylene;
polyethyleneimine; poly(etherether)ketone; polyoxymethylene (POM);
polyvinylphenol; polylactides; polymethacrylimide (PMI);
polyalkenesulfone (PAS); polyhydroxyethylmethacrylate;
polydimethylsiloxane; polyacrylamide; polyimide; co-block-polymers;
and Eupergit.RTM.. Photoresists, polymerized Langmuir-Blodgett
films, and LIGA structures may also serve as substrates in the
present invention. The preferred substrates for the array comprise
silicon, silica, glass, or a polymer.
[0428] In one embodiment of the invention array, the patches
further comprise a monolayer on the surface of the substrate and
the proteins of the patches are unmobilized on the monolayer. The
monolayer is preferably a self-assembling monolayer. This monolayer
may optionally comprise molecules of the formula X--R--Y, wherein R
is a spacer, X is a functional group that binds R to the surface,
and Y is a functional group for binding proteins onto the
monolayer.
[0429] A variety of chemical moieties may function as monolayers in
the array of the present invention. However, three major classes of
monolayer formation are preferably used to expose high densities of
bioreactive omega-functionalities on the patches of the arrays (i)
alkysiloxane monolayers ("silanes") on hydroxylated surfaces (as
taught in, for, example, U.S. Pat. No. 5,405,766, PCT Publication
WO 96/38.726, U.S. Pat. No. 5,412,087, and U.S. Pat. No.
5,688,642); (ii) allyl-thiol/dialkyldisu-lfide monolayers on noble
metals (preferably Au(111)) (as, for example, described in Allara
et al., U.S. Pat. No. 4,690,715; Bamdad et al., U.S. Pat. No.
5,620,850, Wagner et al., Biophysical Journal, 1996, 70:2052-2066);
and (iii) alkyl monolayer formation on oxide-free passivated
silicon (as taught in, for example, Linford et al., J. Am. Chem.
Soc., 1995, 117:3145-3155, Wagner et al., Journal of structural
Biology, 1997, 119:189-201, U.S. Pat. No. 5,429,708). One of
ordinary skill in the art, however, will recognize that many
possible moieties may be substituted for X, R, and/or Y, dependent
primarily upon the choice of substrate, coating, and affinity tag.
Many examples of monolayers are described in Ulman, An Introduction
to Ultrathin Organic Films: From Langmuir-Blodgett to Self
Assembly, Academic press (1991).
[0430] An array of the present invention may optionally further
comprise a coating between the substrate and the monolayer of its
patches. This coating may either be formed on the substrate or
applied to the substrate. The substrate can be modified with a
coating by using thin-film technology based on either physical
vapor deposition (PVD) or plasma-enhanced chemical vapor deposition
(PECVD). Alternatively, plasma exposure can be used to directly
activate the substrate. For instance, plasma etch procedures can be
used to oxidize a polymeric surface (i.e. polystyrene or
polyethylene to expose polar functionalities such as hydroxyls,
carboxylic acids, aldehydes and the like).
[0431] The coating may comprise a metal film. Possible metal films
include aluminum, chromium, titanium, nickel stainless steel zinc,
lead, iron, magnesium, manganese, cadmium, tungsten, cobalt, and
alloys or oxides thereof. In a preferred embodiment, the metal film
is a noble metal film. Noble metals that may be used for a coating
include, but are not limited to, gold, platinum, silver, copper,
and palladium. In another embodiment, the coating comprises gold or
a gold alloy. Electron-beam evaporation may be used to provide a
thin coating of gold on the surface. In yet a further embodiment,
the metal film is from about 50 nm to about 500 nm in
thickness.
[0432] In alternative embodiments, the coating comprises a
composition selected from the group consisting of silicon, silicon
oxide, silicon nitride, silicon hydride, indium tin oxide,
magnesium oxide, alumina, glass, hydroxylated surfaces, and a
polymer.
[0433] An array of the present invention is typically comprised of
a collection of addressable elements. Such elements can be
spacially addressable, such as arrays contained within microtiter
plates or printed on planar surfaces where each element is present
at distinct X and Y coordinates. Alternatively, elements can be
addressable based on tags, beads, nanoparticles, or physical
properties. The microarrays can be prepared according to the
methods known to the ordinarily skilled artisan (See for example,
U.S. Pat. No. 5,807,522; Robinson et al. (2002) Nature Medicine
8:295-301; Robinson et al. (2002) 46:885-93). Arrays as used herein
refers to any biologic assay with multiple addressable elements. In
one embodiment the addressable elements are polypeptides (e.g.,
antibodies or fragments thereof) or nucleic acid probes. As used
herein, elements refer to any probe (polypeptide or nucleic acid
based) that can be bound by an organ-specific polypeptide,
polypeptide fragment or transcript encoding such polypeptides, as
set forth in the appended sequence listing and Tables 1-32, 36-45
and 47-79. Molecules can be, but are not limited to, proteins,
polypeptides, peptides, RNA, DNA, lipids, glycosylated molecules,
carbohydrates, polypeptides with phosphorylation modifications, and
polypeptides with citrulline modifications, aptamers, oxidated
molecules, other molecules, and other molecules.
[0434] For the elements described herein, addressibility refers to
the location, position, tags, cleavable tags or markers,
identifiers, spectral properties, electrophoretic properties, or
other physical properties that enable identification of the
element. One example of addressability, also known as coding, is
spatial addressability, where the position of the molecule is
fixed, and that position is correlated with the identity. This type
of spatial array is generally synthesized or spotted onto a planar
substrate, producing, for example, microarrays, where a large
number of different molecules are densely laid out in a small area,
e.g. comprising at least about 400 different sequences per
cm.sup.2, and may be 1000 sequences per cm.sup.2, or as many as
5000 sequences per cm.sup.2, or more. Less dense arrays, such as
may be found in ELISA or RIA plates where wells in a plate each
contain a distinct probe, may comprise from about 96 sequences per
plate, up to about 100 sequences per cm.sup.2, up to the density of
a microarray. Other spatial arrays utilize fiber optics, where
distinct probes are bound to fibers, which can then be formed into
a bundle for binding and analysis. Methods for the manufacture and
use of spatial arrays of polypeptides are known in the art. Recent
articles include Joos et al. (2000) Electrophoresis 21(13):2641-50
describing a microarray-based immunoassay containing serial
dilutions of probes; Roda et al. (2000) Biotechniques 28(3):492-6
describing a system obtained by adapting a commercial ink-jet
printer and used to produce mono- and bidimensional arrays of spots
containing protein on cellulose paper; and Ge (2000) Nucleic Acids
Res 28(2):e3 describing a universal protein array system for
quantitative detection of protein-protein, protein-DNA, protein-RNA
and protein-ligand interactions. See also, Mendoza et al. (1999)
"High-throughput microarray-based enzyme-linked immunosorbent assay
(ELISA)" Biotechniques 27:778-780; and Lueking et al. (1999)
"Protein microarrays for gene expression and antibody screening"
Anal. Biochem. 270:103-111.
[0435] An alternative to this type of spatial coding array is the
use of molecular "tags," where the target probes are attached to a
detectable label, or tag, which provides coded information about
the sequence of the probe. In certain cases these tags can be
cleaved from the element, and subsequently detected to identity the
element. In another embodiment, a set of probes may be synthesized
or attached to a set of coded beads, where each bead is linked to a
distinct probe, and where the beads are themselves coded in a
manner that allows identification of the attached probe. The use of
a multiplexed microsphere set for analysis of clinical samples by
flow cytometry is described in International Patent application no.
97/14028; and Fulton et al. (1997) Clinical Chemistry
43:1749-1756). It is also possible to use other addressable
particles or tags (reviewed in Robinson et al. (2002) Arthritis
Rheumatism 46:885-93).
[0436] In this type of "tag array," where the probe is bound to
beads or microspheres, one may utilize flow cytometry for detection
of binding. For example, microspheres having fluorescence coding
have been described in the art, where the color and level of
fluorescence uniquely identifies a particular microsphere. The
probe is thus covalently attached to a "color coded" object. A
labeled target polypeptide can be detected by flow cytometry, and
the coding on the microsphere used to identify the bound probe
(e.g., immunoglobulin, antigen binding fragments of
immunoglobulins, or ligands).
[0437] One embodiment of an array is an immunoglobulin (e.g.,
antibody or antigen-binding fragment thereof) array. An
immunoglobulin array as used herein, refers to a spatially
separated set of discrete molecular entities capable of binding to
target polypeptides which are arranged in a manner that allows
identification of the polypeptides contained within the sample. In
other embodiments, the array may comprise one or more of proteins,
polypeptides, peptides, RNA, DNA, lipid, glycosylated molecules,
polypeptides with phosphorylation modifications, and polypeptides
with citrulline modifications, aptamers, other molecules, and other
molecules, where different classes of molecules may be combined in
an array.
[0438] Other detection techniques using click chemistry reagents
(Svenson et al., Adv. Drug. Deliv. Rev. 57(15):2106-2129, 2005;
Kolb et al., Drug Discov. Today 8(24):1128-1137, 2003) or
fluorophore related technologies such as that utilized by
Nanostring Technologies and described in US Patent Application
Publication No. 20030013091, incorporated herein by reference. In
short, this aspect is directed at the use of a diverse population
of unique labels for the detection, identification, and direct
quantification of a wide variety of target analytes. In one
embodiment, the invention is directed to detecting nucleic acid
analytes in a complex mixture by first contacting the mixture under
conditions sufficient for hybridization with a plurality of target
specific nucleic acid probes. These target specific nucleic acid
probes, called specifiers, contain a target specific region and a
region containing one or more unique "genedigit" sequences. The
genedigits consist of repeated core element sequences that can be
specifically bound by a complementary anti-genedigit sequence which
can contain a unique label. The mixture containing the nucleic acid
analytes and the specifiers is then contacted with a corresponding
plurality of labeled anti-genedigits having a diversity sufficient
to uniquely hybridize to genedigits within the specifiers. This
allows the unique detection of a hybridized complex between
analytes in the mixture and specifiers with unique labels.
[0439] The present invention also provides utilizing the
organ-specific sequences disclosed herein to detect and quantify
analytes in a mixture by generating a diverse population of
uniquely labeled probes, contacting a mixture with these probes,
and detecting the complexes that result from hybridization of
probes to analytes in the mixture. This technology may be applied
in a variety of ways, including identifying and quantifying the
expression of genes in normal and diseased cells, as well as aiding
in the discovery of new drug and diagnostic targets.
[0440] The first step in this process involves producing a diverse
population of uniquely labeled nucleic acid probes. This includes
synthesizing a diverse population of target specific nucleic acid
probes each having a different specifier; synthesizing a population
of anti-genedigits capable of specifically binding to the gene
digit of the probe and each having a unique label; and hybridizing
the target nucleic acid probes to the anti-genedigits, thereby
producing a population of uniquely labeled probes. Since a
specifier may contain one or several genedigits the methods herein
may use multiple unique labels may be available to bind analytes in
a mixture. Thus, a large population of specifiers can be
synthesized that contain several combinations of genedigits in
order to label multiple analytes in a mixture. Conversely, in order
to label one or a few analytes in a mixture, a specifier may be
synthesized that contains one or a few genedigits.
[0441] Accordingly, using such genedigits, one can detect an
analyte such as a nucleic acid analyte (such as polypeptides or
transcripts encoding the same from a tissue sample or a sample from
a biological sample such as whole blood) by contacting a mixture of
analytes with a population of uniquely labeled probes, under
conditions sufficient for hybridization. Following this
hybridization, the signals are measured that result from one or
more target specific probes bound to an analyte; wherein the signal
uniquely identifies the analyte.
[0442] The present invention provides a diverse population of
uniquely labeled probes in which a target specific nucleic acid
contains a nucleic acid bound to a unique label. In addition, the
invention provides a diverse population of uniquely labeled probes
containing two attached populations of nucleic acids, one
population of nucleic acids containing thirty or more target
specific nucleic acid probes, and a second population of nucleic
acids containing a nucleic acid bound by a unique label.
[0443] A target specific probe is intended to mean an agent that
binds to the target analyte selectively. This agent will bind with
preferential affinity toward the target while showing little to no
detectable cross-reactivity toward other molecules.
[0444] The target analyte can be any type of macromolecule,
including a nucleic acid, a protein or even a small molecule drug.
For example, a target can be a nucleic acid that is recognized and
bound specifically by a complementary nucleic acid including for
example, an oligonucleotide or a PCR product, or a non-natural
nucleic acid such as a locked nucleic acid (LNA) or a peptide
nucleic acid (PNA). In addition, a target can be a peptide that is
bound by a nucleic acid. For example, a DNA binding domain of a
transcription factor can bind specifically to a particular nucleic
acid sequence. Another example of a peptide that can be bound by a
nucleic acid is a peptide that can be bound by an aptamer. Aptamers
are nucleic acid sequences that have three dimensional structures
capable of binding small molecular targets including metal ions,
organic dyes, drugs, amino acids, co-factors, aminoglycosides,
antibiotics, nucleotide base analogs, nucleotides and peptides
(Jayasena, S. D., Clinical Chemistry 45:9, 1628-1650, (1999))
incorporated herein by reference. Further, a target can be a
peptide that is bound by another peptide or an antibody or antibody
fragment. The binding peptide or antibody can be linked to a
nucleic acid, for example, by the use of known chemistries
including chemical and UV cross-linking agents. In addition, a
peptide can be linked to a nucleic acid through the use of an
aptamer that specifically binds the peptide. Other nucleic acids
can be directly attached to the aptamer or attached through the use
of hybridization. A target molecule can even be a small molecule
that can be bound by an aptamer or a peptide ligand binding
domain.
[0445] The invention further provides a method for detecting a
nucleic acid analyte, by contacting a mixture of nucleic acid
analytes with a population of target specific probes each attached
to a unique label under conditions sufficient for hybridization of
the probes to the target and measuring the resulting signal from
one or more of the target specific probes hybridized to an analyte
where the signal uniquely identifies the analyte.
[0446] The nucleic acid analyte can contain any type of nucleic
acid, including for example, an RNA population or a population of
cDNA copies. The invention provides for at least one target
specific probe for each analyte in a mixture. The invention also
provides for a target specific probe that contains a nucleic acid
bound to a unique label. Furthermore, the invention provides two
attached populations of nucleic acids, one population of nucleic
acids containing a plurality of target specific nucleic acid
probes, and a second population of nucleic acids containing a
nucleic acid bound by a unique label. When the target specific
probes are attached to unique labels, this allows for the unique
identification of the target analytes.
Identification of Unknown Transcripts
[0447] In order to identify organ-specific transcripts, the
resulting annotated transcripts are compared against public and/or
private sequence databases, such as a variety of annotated human
genome sequence databases (e.g., HUPO, Genebank, the EMBL and
Japanese databases and databases generated and compiled from other
normal tissues), to identify those transcripts that are expressed
primarily in the organ of interest but are not expressed in other
organs. As noted elsewhere herein, some expression in organs other
than the organ of interest does not necessarily preclude the use of
a particular transcript in an organ-specific protein set or
diagnostic panel of the present invention.
[0448] In certain embodiments, a particular transcript is
considered to be organ-specific when the number of
transcripts/million as determined by MPSS is 3 copies/million or
greater in the organ of interest but is less than 3 copies/million
in all other organs examined, where, preferably 5, 10, 15, 20 or 25
organs are examined. In another embodiment, a transcript is
considered organ-specific if it is expressed in the organ of
interest at a detectable levels using a standard measurement (e.g.,
microarray analysis, quantitative real-time RT-PCR, MPSS, SBS) in
the organ of interest but is not detectably expressed in other
organs, using appropriate negative and positive controls as would
be familiar to the skilled artisan. In a further embodiment, an
organ-specific transcript is one that is expressed 99% in one organ
and the remaining 1% in one or more other organs examined. (In this
context, total expression across all organs examined is taken as
100%). In certain embodiments, an organ-specific transcript is
expressed at about 50%, 60%, 70%, 80%, 90%, 95% to about 99% in one
organ and wherein the remaining 1%-50% is expressed in one or more
other organs examined. As would be readily recognized by the
skilled artisan upon reading the present disclosure, in certain
embodiments, an organ-specific blood fingerprint can readily be
discerned even if some expression of an organ-specific protein from
a particular organ is detected at some levels in another organ, or
even more than one organ. This is because the fingerprint (e.g.,
the combination of the levels of multiple proteins; the pattern of
the expression levels of multiple markers) itself is unique despite
that the expression levels of one or more individual members of the
fingerprint may not be unique to a particular organ. Thus the
present invention relates to determining the presence or absence of
a disease or condition or stage of disease based on a single marker
or a pattern (e.g., fingerprint) of markers measured concurrently
using any one or more of a variety of methods described herein
(e.g., antibody binding, mass spectrometry, and the like).
[0449] In certain embodiments, the organ-specificity of a
transcript is determined using the algorithms as outlined in
Example 1 or Example 9.
[0450] In further embodiments, organ-specificity can be confirmed
at the protein level using immunohistochemistry (1HC) and/or other
protein measurement techniques known in the art (e.g.,
isotope-coded affinity tags and mass spectrometry, such as
described by Han, D. K., et al., Nat Biotechnol, 19: 946-951,
2001). The Z-test (Man, M. Z., et al., Bioinformatics, 16: 953-959,
2000) or other appropriate statistical tests can be used to
calculate P values for comparison of gene and protein expression
levels between libraries from organs of interest.
[0451] Any of a variety of statistical methods known in the art and
described herein, can be used to evaluate organ-specificity and, as
discussed further herein, define statistical changes in the level
of a particular protein measured between a normal control sample of
blood and a blood sample that is changed from normal. Exemplary
statistical methods include, for example, discriminant analysis,
classification analysis, cluster analysis, analysis of variance
(ANOVA), regression analysis, regression trees, decision trees,
nearest neighbor algorithms, principal components, factor analysis,
multidimensional scaling and other methods of dimensionality
reduction, likelihood models, hypothesis testing, kernel density
estimation and other smoothing techniques, cross-validation and
other methods to guard against overfitting of the data, the
bootstrap and other statistical resampling techniques, artificial
intelligence, including artificial neural networks, machine
learning, data mining, and boosting algorithms, and Bayesian
analysis using prior probability distributions (see e.g., U.S.
Patent Application No. 20020095259).
[0452] Comparisons of the transcripts between databases can be made
using a variety of computer analysis algorithms known in the art.
As such, alignment of sequences for comparison may be conducted by
the local identity algorithm of Smith and Waterman (1981) Add. APL.
Math 2:482, by the identity alignment algorithm of Needleman and
Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity
methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:
2444, by computerized implementations of these algorithms (GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group (GCG), 575 Science Dr.,
Madison, Wis.), or by inspection. As would be understood by the
skilled artisan, many algorithms are available and are continually
being developed. Appropriate algorithms can be chosen based on the
specific needs for the comparisons being made (See also, e.g., J.
A. Cuff, et al., Bioinformatics, 16(2):111-116, 2000; S. F Altschul
and B. W. Erickson. Bulletin of Mathematical Biology,
48(5/6):603-616, 1986; S. F. Altschul and B. W. Erickson. Bulletin
of Mathematical Biology, 48(5/6):633-660, 1986; S. F. Altschul, et
al., J. Mol. Bio., 215:403-410, 1990; K. Bucka-Lassen, et al.,
BIOINFORMATICS, 15(2):122-130, 1999; K.-M. Chao, et al., Bulletin
of Mathematical Biology, 55(3):503-524, 1993; W. M. Fitch and T. F.
Smith. Proceedings of the National Academy of Sciences,
80:1382-1386, 1983; A. D. Gordon. Biometrika, 60:197-200, 1973; 0.
Gotoh. J Mol Biol, 162:705-708, 1982; O. Gotoh. Bulletin of
Mathematical Biology, 52(3):359-373, 1990; X. Huang, et al.,
CABIOS, 6:373-381, 1990; X. Huang and W. Miller. Advances in
Applied Mathematics, 12:337-357, 1991; J. D. Thompson, et al.,
Nucleic Acids Research, 27(13):2682-2690, 1999).
[0453] The organ-specific protein sets may be further characterized
using computational methods to predict localization. In one
embodiment, protein sequences in the RefSeq database are used to
predict protein localization. One of the programs is TMHMM (server
2.0, http colon double slash www dot cbs dot dtu dot
dk/services/TMHMM/), which applies hidden Markov model to predict
protein transmembrane domains and is considered as one of the best
such programs. Another program that can be used in this context is
SignalP (server 3.0, http colon double slash www dot cbs dot dtu
dot dk/services/SignalP/), which applies both artificial neural
network and hidden Markov model to predict the presence and the
location of signal peptide cleavage sites for classical (N-terminus
lead) proteins. The outputs of the two programs can be combined
into protein localization prediction, such as is outlined in Table
33.
[0454] Illustrative computational analyses that can be used for
predicting proteins with signal peptides (classical secretory
proteins) include, but are not limited to the criteria described by
Chen et al., Mamm Genome, 14: 859-865, 2003. In certain
embodiments, such analyses are carried out using prediction
servers, for example SignalP 3.0 server developed by The Center for
Biological Sequence Analysis, Lyngby, Denmark (http colon double
slash www dot cbs dot dtu dot dk/services/SignalP-3.0; see also, J.
D. Bendtsen, et al., J. Mol. Biol., 340:783-795, 2004.) and the
TMHMM2.0 server (see for example A. Krogh, et al., Journal of
Molecular Biology, 305(3):567-580, January 2001; E. L. L.
Sonnhammer, et al., In J. Glasgow, T. Littlejohn, F. Major, R.
Lathrop, D. Sankoff, and C. Sensen, editors, Proceedings of the
Sixth International Conference on Intelligent Systems for Molecular
Biology, pages 175-182, Menlo Park, Calif., 1998. AAAI Press).
Other prediction methods that can be used in the context of the
present invention include those described for example, in S.
Moller, M.D.R. et al., Bioinformatics, 17(7):646-653, July 2001.
Nonclassical secretory secreted proteins (without signal peptides)
can be predicted using, for example, the SecretomeP 1.0 server,
(http colon double slash www dot cbs dot dtu dot
dk/services/SecretomeP-1.0) with an odds ratio score>3.0. Other
methods known in the art are also contemplated herein (e.g., PSORT
(http colon double slash psort dot nibb dot ac dot jp/) and Sigfind
(http colon double slash 139 dot 91 dot 72 dot 10/sigfind/sigfind
dot html).
[0455] As would be recognized by the skilled artisan, while the
organ-specific proteins, the levels of which make up a given normal
or disease-associated fingerprint, need not be isolated, in certain
embodiments, it may be desirable to isolate such proteins (e.g.,
for antibody production or for developing other detection reagents
as described herein). As such, the present invention provides for
isolated organ-specific proteins or fragments or portions thereof
and polynucleotides that encode such proteins. As used herein, the
terms protein and polypeptide are used interchangeably.
Illustrative organ-specific proteins include those provided in the
amino acid sequences set forth in the appended sequence listing.
The terms polypeptide and protein encompass amino acid chains of
any length, including full-length endogenous (i.e., native)
proteins and variants of endogenous polypeptides described herein.
Variants are polypeptides that differ in sequence from the
polypeptides of the present invention only in substitutions,
deletions and/or other modifications, such that either the variants
disease-specific expression patterns are not significantly altered
or the polypeptides remain useful for diagnostics/detection of
organ-specific proteins as described herein. For example,
modifications to the polypeptides of the present invention may be
made in the laboratory to facilitate expression and/or purification
and/or to improve immunogenicity for the generation of appropriate
antibodies and other detection agents. Modified variants (e.g.,
chemically modified) of organ-specific proteins may be useful
herein, (e.g., as standards in mass spectrometry analyses of the
corresponding proteins in the blood, and the like). As such, in
certain embodiments, the biological function of a variant protein
is not relevant for utility in the methods for detection and/or
diagnostics described herein. Polypeptide variants generally
encompassed by the present invention will typically exhibit at
least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity along its
length, to a polypeptide sequence set forth herein. Within a
polypeptide variant, amino acid substitutions are usually made at
no more than 50% of the amino acid residues in the native
polypeptide, and in certain embodiments, at no more than 25% of the
amino acid residues. In certain embodiments, such substitutions are
conservative. A conservative substitution is one in which an amino
acid is substituted for another amino acid that has similar
properties, such that one skilled in the art of peptide chemistry
would expect the secondary structure and hydropathic nature of the
polypeptide to be substantially unchanged. In general, the
following amino acids represent conservative changes: (1) ala, pro,
gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val,
ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp,
his. Thus, a variant may comprise only a portion of a native
polypeptide sequence as provided herein. In addition, or
alternatively, variants may contain additional amino acid sequences
(such as, for example, linkers, tags and/or ligands), usually at
the amino and/or carboxy termini. Such sequences may be used, for
example, to facilitate purification, detection or cellular uptake
of the polypeptide.
[0456] When comparing polypeptide sequences, two sequences are said
to be identical if the sequence of amino acids in the two sequences
is the same when aligned for maximum correspondence, as described
below. Comparisons between two sequences are typically performed by
comparing the sequences over a comparison window to identify and
compare local regions of sequence similarity. A comparison window
as used herein, refers to a segment of at least about 20 contiguous
positions, usually 30 to about 75, 40 to about 50, in which a
sequence may be compared to a reference sequence of the same number
of contiguous positions after the two sequences are optimally
aligned.
[0457] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O. (1978) A
model of evolutionary change in proteins Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure, National Biomedical Research Foundation,
Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990)
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;
Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E.
W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971)
Comb. Theor 11:105; Saitou, N. Nei, M. (1987) Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical
Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman
Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J.
(1983) Proc. Natl. Acad., Sci. USA 80:726-730.
[0458] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman (1981) Add. APL. Math 2:482, by the identity alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity methods of Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[0459] Illustrative examples of algorithms that are suitable for
determining percent sequence identity and sequence similarity
include the BLAST and BLAST 2.0 algorithms, which are described in
Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul
et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and
BLAST 2.0 can be used, for example, to determine percent sequence
identity for the polynucleotides and polypeptides of the invention.
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information.
[0460] An isolated polypeptide is one that is removed from its
original environment. For example, a naturally occurring protein or
polypeptide is isolated if it is separated from some or all of the
coexisting materials in the natural system. In certain embodiments,
such polypeptides are also purified, e.g., are at least about 90%
pure by weight of protein in the preparation, in some embodiments,
at least about 95% pure by weight of protein in the preparation and
in further embodiments, at least about 99% pure by weight of
protein in the preparation.
[0461] In one embodiment of the present invention, a polypeptide
comprises a fusion protein comprising an organ-specific
polypeptide. The present invention further provides fusion proteins
that comprise at least one polypeptide as described herein, as well
as polynucleotides encoding such fusion proteins. The fusion
proteins may comprise multiple polypeptides or portions/variants
thereof, as described herein, and may further comprise one or more
polypeptide segments for facilitating the expression, purification,
detection, and/or activity of the polypeptide(s).
[0462] In certain embodiments, the proteins and/or polynucleotides,
and/or fusion proteins are provided in the form of compositions,
e.g., pharmaceutical compositions, vaccine compositions,
compositions comprising a physiologically acceptable carrier or
excipient. Such compositions may comprise buffers such as neutral
buffered saline, phosphate buffered saline and the like;
carbohydrates such as glucose, mannose, sucrose or dextrans,
mannitol; proteins; polypeptides or amino acids such as glycine;
antioxidants; chelating agents such as EDTA or glutathione;
adjuvants (e.g., aluminum hydroxide); and preservatives.
[0463] In certain embodiments, wash buffer refers to a solution
that may be used to wash and remove unbound material from an
adsorbent surface. Wash buffers typically include salts that may or
may not buffer pH within a specified range, detergents and
optionally may include other ingredients useful in removing
adventitiously associated material from a surface or complex.
[0464] In certain embodiments, elution buffer refers to a solution
capable of dissociating a binding moiety and an associated analyte.
In some circumstances, an elution buffer is capable of disrupting
the interaction between subunits when the subunits are associated
in a complex. As with wash buffers, elution buffers may include
detergents, salt, organic solvents and may be used separately or as
mixtures. Typically, these latter reagents are present at higher
concentrations in an elution buffer than in a wash buffer making
the elution buffer more disruptive to molecular interactions. This
ability to disrupt molecular interactions is termed "stringency,"
with elution buffers having greater stringency that wash
buffers.
[0465] In general, organ-specific polypeptides and polynucleotides
encoding such polypeptides as described herein, may be prepared
using any of a variety of techniques that are well known in the
art. For example, a polynucleotide encoding an organ-specific
protein may be prepared by amplification from a suitable cDNA or
genomic library using, for example, polymerase chain reaction (PCR)
or hybridization techniques. Libraries may generally be prepared
and screened using methods well known to those of ordinary skill in
the art, such as those described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold
Spring Harbor, N.Y., 1989. cDNA libraries may be prepared from any
of a variety of organs, tissues, cells, as described herein. Other
libraries that may be employed will be apparent to those of
ordinary skill in the art upon reading the present disclosure.
Primers for use in amplification may be readily designed based on
the polynucleotide sequences encoding organ-specific polypeptides
as provided herein, for example, using programs such as the PRIMER3
program (see website: http colon double slash www dash genome dot
wi dot mit dot edu slash cgi dash bin slash primer slash primer3
www dot cgi).
[0466] Polynucleotides encoding the organ-specific polypeptides as
described herein are also provided by the present invention.
Polynucleotides of the present invention may comprise a native
sequence (i.e., an endogenous polynucleotide, for instance, a
native or non-artificially engineered or naturally occurring gene
as provided herein) encoding an organ-specific protein, an
alternate form of such a sequence, or a portion or splice variant
thereof or may comprise a variant of such a sequence.
Polynucleotide variants may contain one or more substitutions,
additions, deletions and/or insertions such that the polynucleotide
encodes a polypeptide useful in the methods described herein, such
as for the detection of organ-specific proteins (e.g., wherein said
polynucleotide variants encode polypeptides that can be used to
generate detection reagents as described herein that specifically
bind to an organ-specific protein). In certain embodiments,
variants exhibit at least about 70% identity, and in other
embodiments, exhibit at least about 80%, 85%, 86%, 87%, 88%, 89%,
identity and in yet further embodiments, at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a
polynucleotide sequence that encodes a native organ-specific
polypeptide or an alternate form or a portion thereof. Illustrative
polynucleotides of the present invention comprise the
polynucleotides of set forth in the sequence listing attached
hereto. The percent identity may be readily determined by comparing
sequences using computer algorithms well known to those having
ordinary skill in the art and described herein.
[0467] A polynucleotide as used herein may be single-stranded
(coding or antisense) or double-stranded, and may be DNA (genomic,
cDNA or synthetic) or RNA molecules. Thus, within the context of
the present invention, a polynucleotide encoding a polypeptide may
also be a gene. A gene is a segment of DNA involved in producing a
polypeptide chain; it includes regions preceding and following the
coding region (leader and trailer) as well as intervening sequences
(introns) between individual coding segments (exons). Additional
coding or non-coding sequences may, but need not, be present within
a polynucleotide of the present invention, and a polynucleotide
may, but need not, be linked to other molecules and/or support
materials. An isolated polynucleotide, as used herein, means that a
polynucleotide is substantially away from other coding sequences,
and that the DNA molecule does not contain large portions of
unrelated coding DNA, such as large chromosomal fragments or other
functional genes or polypeptide coding regions. Of course, this
refers to the DNA molecule as originally isolated, and does not
exclude genes or coding regions later added to the segment using
recombinant techniques known to the skilled artisan.
Polynucleotides that are complementary to the polynucleotides
described herein, or that have substantial identity to a sequence
complementary to a polynucleotide as described herein are also
within the scope of the present invention. Substantial identity, as
used herein refers to polynucleotides that exhibit at least about
70% identity, and in certain embodiments, at least about 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity to a polynucleotide sequence that encodes a native
organ-specific polypeptide as described herein. Substantial
identity can also refer to polynucleotides that are capable of
hybridizing under stringent conditions to a polynucleotide
complementary to a polynucleotide encoding an organ-specific
protein. Suitable hybridization conditions include prewashing in a
solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0);
hybridizing at 50-65.degree. C., 5.times.SSC, overnight; followed
by washing twice at 65.degree. C. for 20 minutes with each of
2.times., 0.5.times. and 0.2.times.SSC containing 0.1% SDS.
Nucleotide sequences that, because of code degeneracy, encode a
polypeptide encoded by any of the above sequences are also
encompassed by the present invention. Lastly, it should be
understood by the skilled artisan that RNA as well as cDNA derived
therefrom as well as the coding and non-coding strands may also be
utilized in the methods or as panels described herein in the place
of proteins or antibodies thereto.
Normal Serum Organ-Specific Protein Sets
[0468] A normal serum organ-specific protein set comprises the
subset of proteins from an organ-specific protein set that are
detected in normal serum. Identification of organ-specific proteins
from a given organ-specific protein set that are found in normal
serum can be carried out using a variety of methods known in the
art. For example, antibodies specific for the proteins can be used
to measure the presence of the protein in blood/serum/plasma or
tissue sample/biopsy by a variety of immunoaffinity based
techniques (e.g., immunoblot, Western analysis,
immunoprecipitation, ELISA). Antibodies specific for the proteins
described herein may be commercially available through any of a
number of sources known to the skilled artisan or may be generated
using techniques known in the art and described herein (See, e.g.,
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, 1988).
[0469] As an alternative, aptamers (short DNA or RNA fragments with
binding complementarily to the proteins of interest) may be used in
assays similar to those described for antibodies (see for example,
Biotechniques. 2001 February; 30(2):290-2, 294-5; Clinical
Chemistry. 1999; 45:1628-1650). In this regard, an aptamer may be
selected for specific binding properties and may be used in a
similar manner to an antibody in a variety of appropriate binding
assays known to the skilled artisan and described herein In
addition, antibodies or aptamers may be used in connection with
nanowires to create highly sensitive detections systems (see e.g.,
J. Heath et al., Science. 2004 Dec. 17; 306(5704):2055-6). In
further embodiments, mass spectrometry-based methods can be used to
confirm the presence of a particular protein in the blood.
[0470] A variety of mass spectrometry systems can be employed in
the methods of the invention for identifying and/or quantifying
organ-specific proteins in blood. Mass analyzers with high mass
accuracy, high sensitivity and high resolution include, but are not
limited to, ion trap, triple quadrupole, and time-of-flight,
quadrupole time-of-flight mass spectrometers and Fourier transform
ion cyclotron mass analyzers (FT-ICR-MS). Mass spectrometers are
typically equipped with matrix-assisted laser desorption (MALDI)
and electrospray ionization (ESI) ion sources, although other
methods of peptide ionization can also be used. In ion trap MS,
analytes are ionized by ESI or MALDI and then put into an ion trap.
Trapped ions can then be separately analyzed by MS upon selective
release from the ion trap. Organ-specific proteins can be analyzed,
for example, by single stage mass spectrometry with a MALDI-TOF or
ESI-TOF system. Methods of mass spectrometry analysis are well
known to those skilled in the art (see, for example, Yates, J. Mass
Spect. (1998) 33:1-19; Kinter and Sherman, Protein Sequencing and
Identification Using Tandem Mass Spectrometry, John Wiley &
Sons, New York (2000); Aebersold and Goodlett, Chem. Rev. (2001)
101:269-295; Banez et al, Curr Opin Urol (2005) 15:151-156). For
high resolution protein separation, liquid chromatography ESI-MS/MS
or automated LC-MS/MS, which utilizes capillary reverse phase
chromatography as the separation method, can be used (Yates et al.,
Methods Mol. Biol. (1999) 112:553-569).
[0471] In another embodiment, organ-specific proteins may be
detected and analyzed by immunoaffinity based assays such as
ELISAs, Western blots, and radioimmunoassays. Other methods useful
in this context include isotope-coded affinity tag (ICAT) followed
by multidimensional chromatography and MS/MS. The procedures
described herein for analysis of blood can be modified and adapted
to make use of microfluidics and nanotechnology in order to
miniaturize, parallelize, integrate and automate diagnostic
procedures (see e.g., L. Hood, et al., Science (2004) 306:640-643;
R. H. Carlson, et al., Phys. Rev. Lett. (1997) 79:2149; A. Y. Fu,
et al., Anal. Chem. (2002) 74:2451; J. W. Hong, et al., Nature
Biotechnol. (2004) 22:435; A. G. Hadd, et al., Anal. Chem. (1997)
69:3407; I. Karube, et al., Ann. N.Y. Acad. Sci. (1995) 750:101; L.
C. Waters et al., Anal. Chem. (1998) 70:158; J. Fritz et al.,
Science (2000) 288, 316).
[0472] The levels of organ-specific proteins in blood can also be
measured using any one or more methods such as nucleic acid based
or polypeptide/peptide based microarrays.
[0473] Methods for measuring organ-specific protein levels from
blood/serum/plasma include, but are not limited to, immunoaffinity
based assays such as ELISAs, Western blots, and radioimmunoassays,
fluorescence activated cell sorting (FACS) and mass spectrometry
based methods (matrix-assisted laser desorption ionization (MALDI),
MALDI-Time-of-Flight (TOF), Tandem MS (MS/MS), electrospray
ionization (ESI), Surface Enhanced Laser Desorption Ionization
(SELDI)-TOF MS (Xiao, et al., Mol and Cell Endocrinology 230:95-106
(2005), liquid chromatography (LC)-MS/MS). Other methods useful in
proteomic analysis include 2-D Difference Gel Electrophoresis
(DIGE), and protein arrays (see e.g., Unlu et al., Electrophoresis
18:2071 (1997); Tonge et al, Proteomics 1:377 (2001); Macbeath et
al., Science 289:1760 (2000); Walter et al., Trends in Molecular
Medicine 8:250 (2002).
[0474] In one embodiment, the organ-specific proteins that are
being measured are glycosylated. Thus, in certain embodiments, the
invention contemplates the use of protein glycocapture methods for
preparing proteins for analysis. Protein glycosylation is a very
common post-translational modification. In particular, N-linked
glycosylation is common in proteins that move to extracellular
environments. These include proteins on the extracellular side of
the plasma membrane, secreted proteins and proteins contained in
body fluids. Body fluids include, but are not limited to,
cerebrospinal fluid, blood serum, urine, breast milk, saliva,
pancreatic juice, peritoneal, lacrimal, reproductive, intraocular,
digestive, respiratory, pleural, pericardial, lymphatic, urine,
intracellular and extracellular fluids, and neural fluids. This
list is for illustrative purposes and it is not meant to be
limiting. (Zhang et al., Nat Biotechnol 6:660, (2003)).
Glycoproteins are isolated from any of a variety of tissue samples
or plasma using methods as described in US Patent Application No.
20040023306. After isolating glycopolypeptides from a sample and
cleaving the glycopolypeptide into fragments, the glycopeptide
fragments released from the solid support and the released
glycopeptide fragments are identified and/or quantified. A
particularly useful method for analysis of the released
glycopeptide fragments is mass spectrometry. For high resolution
polypeptide fragment separation, liquid chromatography ESI-MS/MS or
automated LC-MS/MS, which utilizes capillary reverse phase
chromatography as the separation method, can be used (Yates et al.,
Methods Mol. Biol. 112:553-569 (1999)). Data dependent
collision-induced dissociation (CID) with dynamic exclusion can
also be used as the mass spectrometric method (Goodlett, et al.,
Anal. Chem. 72:1112-1118 (2000)). Once a peptide is analyzed by
MS/MS, the resulting CID spectrum can be compared to databases for
the determination of the identity of the isolated glycopeptide.
Methods for protein identification using single peptides has been
described previously (Aebersold and Goodlett, Chem. Rev.
101:269-295 (2001); Yates, J. Mass Spec. 33:1-19 (1998).
[0475] In one embodiment, normal, healthy blood samples are
collected from healthy subjects, proteins present in the blood are
identified using, for example, mass spectrometry, and the proteins
identified in this manner are compared to the organ-specific
proteins provided in Tables 1-32, 36-45 and 47-79 using any of a
variety of computational methods readily known in the art.
[0476] Normal serum organ-specific proteins are generally
identified from a sample of blood collected from a subject using
accepted techniques. In one embodiment, blood samples are collected
in evacuated serum separator tubes. In another embodiment, blood
may be collected in blood collection tubes that contain any
anti-coagulant. Illustrative anticoagulants include
ethylenediaminetetraacetic acid (EDTA) and lithium heparin.
However, any method of blood sample or other bodily fluid or
biological/tissue sample collection and storage is contemplated
herein. In particular blood may be collected by any portal
including the finger, foot, intravenous lines, and portable
catheter lines. In one embodiment, blood is centrifuged and the
serum layer that separates from the red cells is collected for
analysis. In another embodiment, whole blood or plasma is used for
analysis.
[0477] In certain embodiments a normal blood sample is obtained
from human serum recovered from whole blood donations from an
FDA-approved clinical source. In this embodiment, the normal,
healthy donor hematocrit is between the range of 38% and 55%, the
donor weight is over 110 pounds, the donor age is between 18 and 65
years old, the donor blood pressure is in the range of 90-180 mmHg
(systolic) and 50-100 mmHg (diastolic), the arms and general
appearance of the donor are free of needle marks and any mark
signifying risky behavior. The donor pulse should be between 50
bpm-100 bpm, the temperature of the donor should be between 97 and
99.5 degrees. The donor does not have diseases including, but not
limited to chest pain, heart disease or lung disease including
tuberculosis, cancer, skin disease, any blood disease, or bleeding
problems, yellow jaundice, liver disease, hepatitis or a positive
test for hepatitis. The donor has not had close contact with
hepatitis in the past 12 months nor has the donor ever received
pituitary growth hormones.
[0478] In certain embodiments, disease free blood is as follows:
the donor has not made a donation of blood within the previous 8
weeks, the donor has not had a fever with headache within one week
from the date of donation, the donor has not donated a double unit
of red cells using an aphaeresis machine within the previous 16
weeks, the donor is not ill with Severe Acute Respiratory Syndrome
(SARS), nor has the donor had close contact with someone with SARS,
nor has the donor visited (SARS) affected areas. The donor has had
no sexual contact with anyone who has HIV/AIDS or has had a
positive test for the HIV/AIDS virus, and does not have syphilis or
gonorrhea. From 1977 to present, the donor never received money,
drugs, or other payment for sex, male donors have never had sexual
contact with another male, donors have not had a positive test for
the HIV/AIDS virus, donors have not used needles to take drugs,
steroids, or anything not prescribed by a physician, donors have
not used clotting factor concentrates, donors have not had sexual
contact with anyone who was born in or lived in Africa, or traveled
to Africa.
[0479] Thus, the present invention provides the normal serum level
of components that make up a normal serum organ-specific protein
set. This level is an average of the levels of a given component
measured in a statistically large number of blood samples from
normal, healthy individuals. Thus, a "predetermined normal level"
is a statistical range of normal and is also referred to herein as
"predetermined normal range". The normal levels or range of levels
in the blood for each component are determined by measuring the
level of protein in the blood using any of a variety of techniques
known in the art and described herein in a sufficient number of
blood samples from normal, healthy individuals to determine the
standard deviation (SD) with statistically meaningful accuracy.
[0480] As would be recognized by the skilled artisan upon reading
the present disclosure, in determining the normal serum level of a
particular component of an organ-specific protein set, general
biological data is considered and compared, including, for example,
gender, time of day of blood sampling, fasting or after food
intake, age, race, environment and/or polymorphisms. Biological
data may also include data concerning the height, growth rate,
cardiovascular status, reproductive status (pre-pubertal, pubertal,
post-pubertal, pre-menopausal, menopausal, post-menopausal,
fertile, infertile), body fat percentage, and body fat
distribution. This list of individual differences that can be
measured is exemplary and additional biological data is
contemplated.
[0481] Thus, the levels of the components that make up a normal
serum organ-specific protein set are determined. Normal
organ-specific blood fingerprints comprise a data set comprising
determined levels in blood from normal, healthy individuals of one,
two, three, four, five, six, seven, eight, nine, ten, or more
components of a normal serum organ-specific protein set. The normal
levels in the blood for each component included in a fingerprint
are determined by measuring the level of protein in the blood using
any of a variety of techniques known in the art and described
herein, in a sufficient number of blood samples from normal,
healthy individuals to determine the standard deviation (SD) with
statistically meaningful accuracy. Thus, as would be recognized by
one of skill in the art, a determined normal level is defined by
averaging the level of protein measured in a statistically large
number of blood samples from normal, healthy individuals and
thereby defining a statistical range of normal. A normal
organ-specific blood fingerprint comprises the determined levels in
normal, healthy blood of N members of a normal serum organ-specific
protein set wherein N is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
or more members up to the total number of members in a given normal
serum organ-specific protein set. In certain embodiments, a normal
organ-specific blood fingerprint comprises the determined levels in
normal, healthy blood of at least two components of a normal serum
organ-specific protein set. In other embodiments, a normal
organ-specific blood fingerprint comprises the determined levels in
normal, healthy blood of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20 components of a normal serum
organ-specific protein set. In yet further embodiments, a normal
control would be run at the time of the assay such that only the
presence of a normal sample and the test sample would be necessary
and the specific differences between the test sample and the normal
sample would then be delineated based upon the panels provided
herein.
[0482] As would be understood by the skilled artisan upon reading
the present disclosure, the subset of proteins from the
organ-specific protein set that are found in blood may comprise
proteins that are predicted to be secreted, anchored,
transmembrane, or other/intracellular proteins. In this regard a
variety of methods as described herein can be used for predicting
and defining protein localization. As would be recognized by the
skilled artisan, anchored, transmembrane and intracellular proteins
may be detected in the blood for a variety of reasons. For example,
the attachment linkages of anchored proteins may be cleaved by
enzymes or by proteases and thereby be identified in the blood or
biological fluids as an anchored protein. Anchored and
transmembrane organ-specific proteins may also be shed into the
blood. Further, organ-specific proteins that are predicted to be
localized intracellularly may be leaked or excreted into the blood.
In specific embodiments of the present invention, panels and
detection methods may comprise components from or that detect only
organ-specific secreted proteins or transcripts thereof or
components that are leaked, excreted or shed, but not normally
secreted by use of a secretion signal or by means of an alternative
secretion method such as leaderless proteins (e.g., FGF-1, FGF-2,
IL-1.alpha., IL-1.beta., aldose reductase, PD-ECGF, CNTF,
prothymosin .alpha., parathymosin, galectin-1, Factor XIIIa,
ATL-derived factor, annexin-1, transglutaminase, mammary-derived
growth inhibitor, macrophage migration inhibitory factor (MIF), HIV
tat, ATP synthase, aminoacyl-tRNA synthetase, EMAP, rhodanase,
thioredoxin-like protein, and others.
[0483] In certain embodiments, the ability to detect an
organ-specific protein in blood may be hampered due to sensitivity
or other issues. As such, the present invention contemplates
detection of organ-specific proteins from any of a variety of
tissue sources and bodily fluids. Thus, organ-specific proteins can
be measured from biopsy samples from normal or diseased organ or
any bodily fluid, such as, but not limited to, cerebrospinal fluid,
blood serum, urine, breast milk, saliva, pancreatic juice,
peritoneal, lacrimal, reproductive, intraocular, digestive,
respiratory, pleural, pericardial, lymphatic, urine, intracellular
and extracellular fluids, and neural fluids. The present invention
also contemplates detection of organ-specific proteins at the
transcript level from any of these tissue sources using
polynucleotide-based detection methods known in the art and
described herein.
Diagnostic/Prognostic Panels
[0484] The normal serum organ-specific protein sets defined herein
and the predetermined normal levels of the components that make up
the organ-specific protein sets (e.g., the database of
predetermined normal serum levels of organ-specific proteins) can
be used as a baseline against which one can determine any
perturbation of the normal state. Perturbation of the normal
biological state is identified by measuring levels of
organ-specific proteins from a patient and comparing the measured
levels against the predetermined normal levels. Any level that is
statistically significantly altered from the normal level (i.e.,
any level from the disease sample that is outside (either above or
below) the predetermined normal range) indicates a perturbation of
normal and thus, the presence of disease (or effect of a drug or
environmental agent, etc.). In this way, the predetermined normal
levels of normal serum organ-specific proteins are also used to
identify and define disease-associated blood fingerprints. Such
sets or panels typically comprise proteins or nucleic acid
molecules that are organ-specific, but that may be found in a
bodily fluid or tissue sample. In certain embodiments the present
methods, panels, and sets are directed to either collective sets or
individual sets of organ-specific proteins that can be detected in
a bodily fluid and are secreted, leaked, excreted or shed. In
certain specific embodiments, the present invention is directed to
sets of proteins (including antibodies and fragments that bind
thereto) that are secreted or the nucleic acid molecules that
encode the same or nucleic acid probes that bind thereto. As used
herein, a panel may comprise less than the entire set of sequences
defined in the tables attached hereto for a given organ. For
example, as can be readily appreciated by the skilled artisan, 1
transcript or protein of each organ may be enough to generally
monitor the health of an organ. However, increasing the number of
probes targeting the component (nucleic acid or polypeptide), while
not necessary will add specificity and sensitivity to the assay.
Accordingly, in certain aspects at least 5 probes per organ set for
organ-specific components will be present in the panel, in other
aspects at least 10 probes per organ set will be present, yet in
others there may be 20, 30, 40, 50 or more probes present per organ
set. In certain embodiments, probes per set may include 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100,
110 or any integer value therebetween.
[0485] Thus, the present invention provides panels for detecting
and measuring the level of organ-specific proteins in blood that
can be used in a variety of diagnostic settings. As used herein and
discussed further below, "diagnostic panel or prognostic panel" is
meant to encompass panels, arrays, mixtures, and kits that may
comprise detection reagents or probes specific to an organ specific
component or a control (control nucleic acid or polypeptide
sequences may or may not be a component of an organ specific set)
and any of a variety of associated buffers, solutions, appropriate
negative and positive controls, instruction sets, and the like. A
"detection reagent" as used herein is meant to refer to any agent
that that associates or binds directly or indirectly to a molecule
in the test sample. In certain embodiments, a detection reagent may
comprise antibodies (or fragments thereof) either with a secondary
detection reagent attached thereto or without, nucleic acid probes,
aptamers, click reagents, etc. Further, a "panel" may comprise
panels, arrays, mixtures, kits, or other arrangements of proteins,
antibodies or fragments thereof to organ-specific proteins, nucleic
acid molecules encoding organ-specific proteins, nucleic acid
probes to that hybridize to organ-specific nucleic acid sequences.
Moreover, a panel may be derived from only one organ or two or more
organs. In certain embodiments, organs that comprise a certain
system such as the cardiovascular or central nervous system may be
grouped together.
[0486] The present invention provides panels for detecting the
organ-specific blood proteins at any given time in a subject.
Examples of subjects include humans, monkeys, apes, dogs, cats,
mice, rats, fish, zebra fish, birds, horses, pigs, cows, sheep,
goats, chickens, ducks, donkeys, turkeys, peacocks, chinchillas,
ferrets, gerbils, rabbits, guinea pigs, hamsters and transgenic
species thereof. Further subjects contemplated herein include, but
are not limited to, reptiles and amphibians, e.g., lizards, snakes,
turtles, frogs, toads, salamanders, and newts and transgenic
species thereof.
[0487] The panels are comprised of a plurality (e.g., at least two)
of detection reagents that each specifically detects a protein (or
transcript), in most embodiments substantially all are
organ-specific but may also comprise non-organic specific reagents
for use as controls or other purposes. In certain aspects the
panels comprise detection reagents that each specifically detects a
protein (or transcript) an organ-specific protein, wherein the
levels of organ-specific proteins taken together form a unique
pattern that defines a fingerprint. In certain embodiments,
detection reagents can be bispecific such that the panel is
comprised of a plurality of bispecific detection reagents that may
specifically detect more than one organ-specific protein. The term
specifically is a term of art that would be readily understood by
the skilled artisan to mean, in this context, that the protein of
interest is detected by the particular detection reagent but other
proteins are not substantially detected. Specificity can be
determined using appropriate positive and negative controls and by
routinely optimizing conditions.
[0488] The diagnostic panels of the present invention comprise
detection reagents wherein each detection reagent is specific for
one protein or transcript of an organ or tissue, but as noted
above, may also comprise controls that are not or may not be
specific to a particular organ/tissue-specific protein or
transcript. In certain embodiments, the detection reagents of a
panel can each be specific for organ-specific proteins from one
organ-specific protein set or from more than one organ-specific
protein set. For example, a particular diagnostic panel may
comprise detection reagents that detect one, two, three, four,
five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty,
twenty-one, twenty-two, twenty-three, twenty-four, twenty-five,
twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty,
thirty-one, thirty-two, thirty-three, thirty-four, thirty-five,
thirty-six, thirty-seven, thirty-eight, thirty-nine, forty,
forty-one, forty-two, forty-three, forty-four, forty-five,
forty-six, forty-seven, forty-eight, forty-nine, fifty, sixty,
seventy, eighty, ninety, one-hundred or more prostate-specific
proteins, such as those provided in Table 21, or a diagnostic panel
may comprise detection reagents that detect one or more
bladder-specific proteins and one or more kidney-specific
proteins.
[0489] In specific embodiments, and as noted above, diagnostic or
prognostic panels may include panels having reagents (e.g., probes)
that bind organ-specific proteins or transcripts from one or more
organs. To this end, it is envisioned that a panel such as an
microarray can have placed thereon multiple protein or nucleic acid
probes which specifically bind the organ-specific protein or
transcript identified by the methods herein and/or expressly
recited in the tables and sequence listing provided herewith.
Further, such an array may have placed thereon probes specific for
one, two, three, four, five, six, seven, eight, nine, ten, eleven,
twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,
nineteen, twenty or more organs. Further, each organ could be
represented with one, two, three, four, five, six, seven, eight,
nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,
seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two,
twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven,
twenty-eight, twenty-nine, thirty, thirty-one, thirty-two,
thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven,
thirty-eight, thirty-nine, forty, forty-one, forty-two,
forty-three, forty-four, forty-five, forty-six, forty-seven,
forty-eight, forty-nine, fifty, sixty, seventy, eighty, ninety,
one-hundred or more probes. Moreover, a single array may comprise
organs associated with a particular bodily system, such as, the
reproductive system (ovaries, uterus, etc.), cardiovascular system
(heart, lungs, etc.), respiratory system, nervous system, endocrine
system, skeletal system, etc. Lastly, it is contemplated that one
could utilize a general health panel that screens one or more
organ/tissue specific proteins or transcripts from nearly every
organ and if an anomaly is noted a follow-up screen with a more
detailed panel comprising additional probes for the anomalous
organ.
[0490] In certain embodiments, the diagnostic panels comprise one
or more detection reagents. In another embodiment, a diagnostic
panel of the invention may comprise two or more detection reagents.
Thus, the diagnostic panels of the invention may comprise a
plurality of detection reagents. As would be recognized by the
skilled artisan, the number of detection reagents on a given panel
would be determined from the number of organ-specific proteins to
be measured. In this regard, the plurality of detection reagents
may be anywhere from 2 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,
150, 160, 170, 180, 190, 200 or more detection reagents each
specific for an organ-specific protein. In specific embodiments,
the panel may comprise for example, 10-50 probes per
organ/tissue/cell type and probe 30-50 organs/tissues or more.
Accordingly, such arrays/panels may comprise 2500 or more probes.
In one embodiment, the panels of the invention comprises at least
3, 4, 5, 6, 7, 8, 9, or 10 detection reagents each specific for one
of the plurality of organ-specific proteins that make up a given
fingerprint. In another embodiment, the panel comprises at least
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 detection reagents each
specific for one of the plurality of organ-specific proteins that
make up a given fingerprint. In a further embodiment, the panel
comprises at least 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
detection reagents each specific for one of the plurality of
organ-specific proteins that make up a given fingerprint. In an
additional embodiment, the panel comprises at least 31, 32, 33, 34,
35, 36, 37, 38, 39, or 40 detection reagents each specific for one
of the plurality of organ-specific proteins that make up a given
fingerprint. In yet a further embodiment, the panel comprises at
least 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 detection reagents
each specific for one of the plurality of organ-specific proteins
that make up a given fingerprint. In an additional embodiment, the
panel comprises at least 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60
detection reagents each specific for one of the plurality of
organ-specific proteins that make up a given fingerprint. In one
embodiment, the panel comprises at least 61, 62, 63, 64, 65, 66,
67, 68, 69, or 70 detection reagents each specific for one of the
plurality of organ-specific proteins that make up a given
fingerprint. In one embodiment, the panel comprises at least 75,
80, 85, 90, 100, 150, 160, 170, 180, 190, 200, or more, detection
reagents each specific for one of the plurality of organ-specific
proteins that make up a given fingerprint.
[0491] In one aspect, the detection reagents specific for the
organ/tissue specific transcripts may be utilized in a
multiparameter analysis method such as a method of classifying a
population by drug responsiveness, comprising: (a) determining a
multidimensional coordinate point representative of the expression
levels of a sample of molecules in a specimen from individuals in a
population of individuals administered a drug; and (b) determining
a drug response-associated reference expression region of a group
of individuals in said population using said multidimensional
coordinate points, thereby classifying said group of individuals
into a drug response reference population. Accordingly, the method
provides a means of determining a comparative expression profile in
an individual by comparing the expression levels of a sample of
molecules in a population of molecules in a specimen from the
individual with a health-associated reference expression region of
the sample of molecules, wherein expression levels within the
health-associated reference expression region indicate a reference
expression profile and wherein expression levels outside the
health-associated reference expression region indicate a perturbed
expression profile. In addition, the method can be used for
diagnosing a disease or a health state in an individual by
comparing the expression level of a sample of molecules in a
specimen from the individual with a health-associated reference
expression region of the sample of molecules. Additionally, the
reagent probes may be used in a method of classifying a population
by drug responsiveness such methods are described in greater detail
in U.S. Patent Application Publication No. 20020095259.
[0492] Panels of the invention comprise N detection reagents
wherein N is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more
detection reagents up to the total number of members in a given
organ-specific protein set that are to be detected. As noted above,
in certain embodiments, it may be desirable to detect proteins from
two or more organ-specific protein sets. Accordingly, the
diagnostic panels of the invention may comprise N detection
reagents wherein N is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
or more detection reagents up to the total number of members in one
or more organ-specific protein sets that are to be detected.
Detection reagents of a given diagnostic panel may detect proteins
from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, or more organ-specific protein sets, such as those provided
in Tables 1-32, 36-45 and 47-79, or normal serum organ-specific
protein sets thereof.
[0493] Organ-specific proteins can be detected and measured using
any of a variety of detection reagents in the context of a variety
of methods for measuring protein levels. Any detection reagent that
can specifically bind to or otherwise detect an organ-specific
protein as described herein is contemplated as a suitable detection
reagent. Illustrative detection reagents include, but are not
limited to antibodies, or antigen-binding fragments thereof, yeast
ScFv, DNA or RNA aptamers, isotope labeled peptides, receptors,
ligands, click reagents, molecular beacons, quantum dots,
microfluidic/nanotechnology measurement devices and the like.
[0494] In one illustrative embodiment, a detection reagent is an
antibody or an antigen-binding fragment thereof. Methods of
producing polyclonal antibodies are well known to those skilled in
the art. Exemplary protocols which may be used are described for
example in Coligan et al., "Current Protocols In Immunology", (John
Wiley & Sons, Inc, 1991 and subsequent updates). Monoclonal
antibodies may be produced using the standard method as described,
for example, by Kohler and Milstein (1975, Nature 256, 495-497), or
by more recent modifications thereof as described, for example, in
Coligan et al., (1991, supra) by immortalizing spleen or other
antibody-producing cells derived from a production species which
has been inoculated with an organ-specific protein of the
invention. In general, antibodies can be produced by cell culture
techniques, including the generation of monoclonal antibodies as
described herein, or via transfection of antibody genes into
suitable bacterial or mammalian cell hosts, in order to allow for
the production of recombinant antibodies. In one technique, an
immunogen comprising the polypeptide is initially injected into any
of a wide variety of mammals (e.g., mice, rats, rabbits, chicken,
sheep or goats). In this step, the polypeptides of this invention
may serve as the immunogen without modification. Alternatively,
particularly for relatively short polypeptides, a superior immune
response may be elicited if the polypeptide is joined to a carrier
protein, such as bovine serum albumin or keyhole limpet hemocyanin.
The immunogen is injected into the animal host, usually according
to a predetermined schedule incorporating one or more booster
immunizations, and the animals are bled periodically. Polyclonal
antibodies specific for the polypeptide may then be purified from
such antisera by, for example, affinity chromatography using the
polypeptide coupled to a suitable solid support.
[0495] In one embodiment, multiple target proteins or peptides are
used in a single immune response to generate multiple useful
detection reagents simultaneously. In one embodiment, the
individual specificities are later separated out.
[0496] In certain embodiments, antibody can be generated by phage
display methods (such as described by Vaughan, T. J., et al., Nat
Biotechnol, 14: 309-314, 1996; and Knappik, A., et al., Mol Biol,
296: 57-86, 2000); ribosomal display (such as described in Hanes,
J., et al., Nat Biotechnol, 18: 1287-1292, 2000), or periplasmic
expression in E. coli (see e.g., Chen, G., et al., Nat Biotechnol,
19: 537-542, 2001.). In further embodiments, antibodies can be
isolated using a yeast surface display library. See e.g., nonimmune
library of 10.sup.9 human antibody scFv fragments as constructed by
Feldhaus, M. J., et al., Nat Biotechnol, 21: 163-170, 2003. There
are several advantages of this yeast surface display compared to
more traditional large nonimmune human antibody repertoires such as
phage display, ribosomal display, and periplasmic expression in E.
coli 1). The yeast library can be amplified 10.sup.19-fold without
measurable loss of clonal diversity and repertoire bias as the
expression is under control of the tightly GAL1/10 promoter and
expansion can be done under non induction conditions; 2)
nanomolar-affinity scFvs can be routinely obtained by magnetic bead
screening and flow-cytometric sorting, thus greatly simplified the
protocol and capacity of antibody screening; 3) with equilibrium
screening, a minimal affinity threshold of the antibodies desired
can be set; 4) the binding properties of the antibodies can be
quantified directly on the yeast surface; 5) multiplex library
screening against multiple antigens simultaneously is possible; and
6) for applications demanding picomolar affinity (e.g. in early
diagnosis), subsequent rapid affinity maturation (Kieke, M. C., et
al., J Mol Biol, 307: 1305-1315, 2001.) can be carried out directly
on yeast clones without further re-cloning and manipulations.
[0497] Monoclonal antibodies specific for an organ-specific
polypeptide of interest may be prepared, for example, using the
technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976,
and improvements thereto. Briefly, these methods involve the
preparation of immortal cell lines capable of producing antibodies
having the desired specificity (i.e., reactivity with the
polypeptide of interest). Such cell lines may be produced, for
example, from spleen cells obtained from an animal immunized as
described above. The spleen cells are then immortalized by, for
example, fusion with a myeloma cell fusion partner, in certain
embodiments, one that is syngeneic with the immunized animal. A
variety of fusion techniques may be employed. For example, the
spleen cells and myeloma cells may be combined with a nonionic
detergent for a few minutes and then plated at low density on a
selective medium that supports the growth of hybrid cells, but not
myeloma cells. An illustrative selection technique uses HAT
(hypoxanthine, aminopterin, thymidine) selection. After a
sufficient time, usually about 1 to 2 weeks, colonies of hybrids
are observed. Single colonies are selected and their culture
supernatants tested for binding activity against the polypeptide.
Hybridomas having high reactivity and specificity are
preferred.
[0498] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the
hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host, such as a mouse. Monoclonal antibodies may then be
harvested from the ascites fluid or the blood. Contaminants may be
removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation, and extraction. The
polypeptides of this invention may be used in the purification
process in, for example, an affinity chromatography step.
[0499] A number of diagnostically useful molecules are known in the
art which comprise antigen-binding sites that are capable of
exhibiting immunological binding properties of an antibody
molecule. The proteolytic enzyme papain preferentially cleaves IgG
molecules to yield several fragments, two of which (the F(ab)
fragments) each comprise a covalent heterodimer that includes an
intact antigen-binding site. The enzyme pepsin is able to cleave
IgG molecules to provide several fragments, including the
F(ab').sub.2 fragment which comprises both antigen-binding sites.
An Fv fragment can be produced by preferential proteolytic cleavage
of an IgM, and on rare occasions IgG or IgA immunoglobulin
molecule. Fv fragments are, however, more commonly derived using
recombinant techniques known in the art. The Fv fragment includes a
non-covalent V.sub.H::V.sub.L heterodimer including an
antigen-binding site which retains much of the antigen recognition
and binding capabilities of the native antibody molecule. Inbar et
al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al.
(1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem
19:4091-4096.
[0500] A single chain Fv (sFv) polypeptide is a covalently linked
V.sub.H::V.sub.L heterodimer which is expressed from a gene fusion
including V.sub.H- and V.sub.L-encoding genes linked by a
peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci.
USA 85(16):5879-5883. A number of methods have been described to
discern chemical structures for converting the naturally aggregated
but chemically separated light and heavy polypeptide chains from an
antibody V region into an sFv molecule which will fold into a three
dimensional structure substantially similar to the structure of an
antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and
5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner
et al.
[0501] Each of the above-described molecules includes a heavy chain
and a light chain CDR set, respectively interposed between a heavy
chain and a light chain FR set which provide support to the CDRS
and define the spatial relationship of the CDRs relative to each
other. As used herein, the term CDR set refers to the three
hypervariable regions of a heavy or light chain V region.
Proceeding from the N-terminus of a heavy or light chain, these
regions are denoted as CDR1, CDR2, and CDR3 respectively. An
antigen-binding site, therefore, includes six CDRs, comprising the
CDR set from each of a heavy and a light chain V region. A
polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3)
is referred to herein as a molecular recognition unit.
Crystallographic analysis of a number of antigen-antibody complexes
has demonstrated that the amino acid residues of CDRs form
extensive contact with bound antigen, wherein the most extensive
antigen contact is with the heavy chain CDR3. Thus, the molecular
recognition units are primarily responsible for the specificity of
an antigen-binding site.
[0502] As used herein, the term FR set refers to the four flanking
amino acid sequences which frame the CDRs of a CDR set of a heavy
or light chain V region. Some FR residues may contact bound
antigen; however, FRs are primarily responsible for folding the V
region into the antigen-binding site, particularly the FR residues
directly adjacent to the CDRS. Within FRs, certain amino residues
and certain structural features are very highly conserved. In this
regard, all V region sequences contain an internal disulfide loop
of around 90 amino acid residues. When the V regions fold into a
binding-site, the CDRs are displayed as projecting loop motifs
which form an antigen-binding surface. It is generally recognized
that there are conserved structural regions of FRs which influence
the folded shape of the CDR loops into certain canonical structures
regardless of the precise CDR amino acid sequence. Further, certain
FR residues are known to participate in non-covalent interdomain
contacts which stabilize the interaction of the antibody heavy and
light chains.
[0503] In certain embodiments the use of click chemistry (e.g.,
click reagents) to anchor on or more probes/reagents specific to an
organ/tissue specific protein or transcript to a detection label or
to an array or other surface (e.g., nanoparticle). While such
chemistries are well known in the art, in short, the chemistries
utilized allow bioconjugation by the formation of triazoles that
readily associate with biological targets, through hydrogen bonding
and dipole interactions. Chemistries such as this are detailed in
the art that is incorporated herein by reference in its entirety
and includes Kolb and Sharpless, DDT, Vol. 8 (24), 1128-1137, 2003;
U.S. Patent Application Publication No. 20050222427.
[0504] The detection reagents of the present invention may comprise
any of a variety of detectable labels or reporter groups. The
invention contemplates the use of any type of detectable label,
including, e.g., visually detectable labels, fluorophores, and
radioactive labels. The detectable label may be incorporated within
or attached, either covalently or non-covalently, to the detection
reagent. Detectable labels or reporter groups may include
radioactive groups, dyes, fluorophores, biotin, colorimetric
substrates, enzymes, or colloidal compounds. Illustrative
detectable labels or reporter groups include but are not limited
to, fluorescein, tetramethyl rhodamine, Texas Red, coumarins,
carbonic anhydrase, urease, horseradish peroxidase, dehydrogenases
and/or colloidal gold or silver. For radioactive groups,
scintillation counting or autoradiographic methods are generally
appropriate for detection. Spectroscopic methods may be used to
detect dyes, luminescent groups and fluorescent groups. Biotin may
be detected using avidin, coupled to a different reporter group
(commonly a radioactive or fluorescent group or an enzyme). Enzyme
reporter groups may generally be detected by the addition of
substrate (generally for a specific period of time), followed by
spectroscopic or other analysis of the reaction products.
[0505] The present invention also contemplates detecting
polynucleotides that encode the organ-specific proteins of the
present invention. Accordingly, detection reagents also include
polynucleotides, oligonucleotide primers and probes that
specifically detect polynucleotides encoding any of the
organ-specific proteins as described herein from any of a variety
of tissue sources. Thus, the present invention contemplates
detection of expression levels by detection of polynucleotides
encoding any of the organ-specific proteins described herein using
any of a variety of known techniques including, for example, PCR,
RT-PCR, quantitative PCR, real-time PCR, northern blot analysis,
and the like. Oligonucleotide primers for amplification of the
polynucleotides encoding organ-specific proteins are within the
scope of the present invention where polynucleotide-based detection
is desired to better detect organ-specific proteins in a diagnostic
assay or kit. Oligonucleotide primers for amplification of the
polynucleotides encoding organ-specific proteins are also within
the scope of the present invention to amplify transcripts in a
biological sample. Many amplification methods are known in the art
such as PCR, RT-PCR, quantitative real-time PCR, and the like. The
PCR conditions used can be optimized in terms of temperature,
annealing times, extension times and number of cycles depending on
the oligonucleotide and the polynucleotide to be amplified. Such
techniques are well known in the art and are described in, for
example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol.,
51:263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989.
Oligonucleotide primers can be anywhere from 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or
30 nucleotides in length. In certain embodiments, the
oligonucleotide primers/probes of the present invention are
typically 35, 40, 45, 50, 55, 60, or more nucleotides in
length.
[0506] The panels of the present invention may be comprised of a
solid phase surface having attached thereto a plurality of
detection reagents each attached at a distinct location. Further in
this regard, the solid phase surface may be of any material,
including, but not limited to, plastic, polycarbonate, polystyrene,
polypropylene, polyethlene, glass, nitrocellulose, dextran, nylon,
metal, silicon and carbon nanowires, nanoparticles that can be made
of a variety of materials and photolithographic materials. In
certain embodiments, the solid phase surface is a chip. In another
embodiment, the solid phase surface may comprise microtiter plates,
beads, membranes, microparticles, the interior surface of a
reaction vessel such as a test tube or other reaction vessel. In
other embodiments the peptides will be fractionated by one or more
one-dimensional columns using size separations, ion exchange or
hydrophobicity properties and, for example, deposited in a MALDI 96
or 384 well plate and then injected into an appropriate mass
spectrometer.
[0507] In one embodiment, the panel is an addressable array. As
such, the addressable array may comprise a plurality of distinct
detection reagents, such as antibodies, aptamers or
oligonucleotides, attached to precise locations on a solid phase
surface, such as a plastic chip. The position of each distinct
detection reagent on the surface is known and therefore
addressable. In one embodiment, the detection reagents are distinct
antibodies that each has specific affinity for one of a plurality
of organ-specific polypeptides.
[0508] In one embodiment, the detection reagents, such as
antibodies, are covalently linked to the solid surface, such as a
plastic chip, for example, through the Fc domains of antibodies. In
another embodiment, antibodies are adsorbed onto the solid surface.
In a further embodiment, the detection reagent, such as an
antibody, is chemically conjugated to the solid surface. In a
further embodiment, the detection reagents are attached to the
solid surface via a linker.
[0509] Methods of constructing protein arrays, including antibody
arrays, are known in the art (see, e.g., U.S. Pat. No. 5,489,678;
U.S. Pat. No. 5,252,743; Blawas and Reichert, 1998, Biomaterials
19:595-609; Firestone et al., 1996, J. Amer. Chem. Soc. 18,
9033-9041; Mooney et al., 1996, Proc. Natl. Acad. Sci. 93,
12287-12291; Pirrung et al, 1996, Bioconjugate Chem. 7, 317-321;
Gao et al, 1995, Biosensors Bioelectron 10, 317-328; Schena et al,
1995, Science 270, 467-470; Lom et al., 1993, J. Neurosci. Methods,
385-397; Pope et al., 1993, Bioconjugate Chem. 4, 116-171; Schramm
et al., 1992, Anal. Biochem. 205, 47-56; Gombotz et al., 1991, J.
Biomed. Mater. Res. 25, 1547-1562; Alarie et al., 1990, Analy.
Chim. Acta 229, 169-176; Owaku et al, 1993, Sensors Actuators B,
13-14, 723-724; Bhatia et al., 1989, Analy. Biochem. 178, 408-413;
Lin et al., 1988, IEEE Trans. Biomed. Engng., 35(6), 466-471).
[0510] In one embodiment, the detection reagents, such as
antibodies or aptamers, are arrayed on a chip comprised of
electronically activated copolymers of a conductive polymer and the
detection reagent. Such arrays are known in the art (see e.g., U.S.
Pat. No. 5,837,859 issued Nov. 17, 1998; PCT publication WO
94/22889 dated Oct. 13, 1994). The arrayed pattern may be computer
generated and stored. The chips may be prepared in advance and
stored appropriately. The antibody array chips can be regenerated
and used repeatedly.
[0511] In certain embodiments, detection with multiple specific
detection reagents is carried out in solution.
[0512] The detection reagents of the present invention may be
provided in a diagnostic kit. As such a diagnostic kit may comprise
any of a variety of appropriate reagents or buffers, enzymes, dyes,
colorimetric or other substrates, and appropriate containers to be
used in any of a variety of detection assays as described herein.
Kits may also comprise one or more positive controls, one or more
negative controls, and a protocol for identification of the
organ-specific proteins of interest using any one of the assays as
described herein.
[0513] In certain embodiments, the detection reagents for a
diagnostic panel are selected such that the level of at least one
of the organ-specific proteins detected by the plurality of
detection reagents in a blood sample from a subject afflicted with
a disease affecting the organ or organs from which the
organ-specific proteins are derived is above or below a
predetermined normal range. In certain embodiments, the detection
reagents for a diagnostic panel are selected such that the level of
at least two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,
eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three,
twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight,
twenty-nine, thirty, thirty-one, thirty-two, thirty-three,
thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight,
thirty-nine, forty, forty-one, forty-two, forty-three, forty-four,
forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty,
sixty, seventy, eighty, ninety, one-hundred or more of the
organ-specific proteins detected by the plurality of detection
reagents in a biological sample (e.g., blood) from a subject
afflicted with a disease affecting the organ or organs from which
the organ-specific proteins are derived is above or below a
predetermined normal range. Thus, the detection reagents for a
diagnostic panel, kit, or array may be selected such that the level
of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
60, 70, 80, 90, 100, 110 or any integer value therebetween, or more
of the organ-specific proteins detected by the plurality of
detection reagents in a blood sample from a subject afflicted with
a disease affecting the organ or organs from which the
organ-specific proteins are derived is above or below a
predetermined normal range.
[0514] The levels and locations of organ-specific proteins may
change as the result of disease. Thus, in certain embodiments, in
vivo imaging techniques can be used to visualize the levels and
locations of organ-specific proteins in bodily fluid. In this
embodiment, exemplary in vivo imaging techniques include, but are
not limited to PET, SPECT (Sharma et al; Journal of Magnetic
Resonance Imaging (2002), 16: 336-351), MALDI (Stoeckli, et al.
Nature Medicine (2001) 7: 493-496), and Fluorescence resonance
energy transfer (FRET) (Seker et al, The Journal of Cell Biology,
160 5, (2003) 629-633).
Methods of Use
[0515] The present invention provides organ-specific protein and
transcript sets and normal serum organ-specific protein and
transcript sets, panels thereof, reagents and probes directed
thereto and methods for use and identifying the same. The present
invention further provides panels, arrays, mixtures, and kits
comprising detection reagents or probes for detecting such
organ-specific proteins or polynucleotides that encode them in
blood, other bodily fluid, and tissue samples such as biopsy
samples from diseased organs.
[0516] It should also be understood that the blood protein and
transcript fingerprints constitute assays for the normal organ and
all the diseases of the organ. Thus all different diseases
affecting such organ either directly or indirectly may be detected
or monitored because each different type of disease arises from
distinct disease-perturbed networks that change the levels of
different combinations of proteins whose synthesis they control.
The present invention is not claiming disease-specific proteins,
rather the fingerprints report the organ status for all different
normal and disease organ conditions.
[0517] The present invention further provides methods of
identifying new drug targets for a disease or indication by
detecting specific up-regulation of a transcript or polypeptide in
a diseased state. In addition, the present invention contemplates
using such targets for imaging or drug targeting such that a probe
to a disease specific protein or transcript may be utilized alone
as a targeting agent or coupled to another therapeutic or
diagnostic imaging agent.
[0518] The present invention also provides defined normal and
disease-associated organ-specific blood fingerprints. As such, the
present invention provides methods of detecting diseases or
following disease progression. The invention further provides
methods for stratifying disease types and for monitoring the
progression of a disease. The present invention also provides for
following responses to therapy, stratifying or qualifying patients
for therapy or a clinical trial, in a variety of disease settings
and methods for detecting the disease state in humans using the
visualization of nanoparticles with appropriate reporter groups and
organ-specific antibodies or aptamers.
[0519] The present invention can be used as a standard screening
test. In this regard, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 60, 70, 80, 90, 100, 110 or any integer value
therebetween or more of the detection reagents specific for the
organ-specific proteins described herein can be used to measure the
level of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60,
70, 80, 90, 100, 110 or any integer value therebetween or more
organ-specific proteins in a blood sample and any statistically
significant deviation from a normal serum organ-specific blood
fingerprint would indicate that disease-related perturbation was
present. Thus, the present invention provides a normal
organ-specific blood fingerprint for any given organ. In certain
embodiments, a normal organ-specific blood fingerprint is
determined by measuring the normal range of levels of the
individual protein members of a fingerprint. Any deviation
therefrom or perturbation of the normal fingerprint that is outside
the standard deviation (normal range) has diagnostic utility (see
also U.S. Patent Application No. 0020095259). As would be
recognized by the skilled artisan, the significance of any
deviation in the levels of (e.g., a significantly altered level of
one or more of) the individual protein members of a fingerprint can
be determined using statistical methods known in the art and
described herein. As noted elsewhere herein, perturbation of the
normal fingerprint can indicate primary disease of the organ being
tested or secondary, indirect affects on that organ resulting from
disease of another organ. Perturbation from normal may also include
the presence of a protein in a sample of a patient being tested for
a perturbed state not present in organ-specific panel (e.g., when
analyzing a certain patient sample such as in the prostate a
protein or transcript not found in the normal prostate panel may
appear in a perturbed sample) may be an indicator of disease.
Further, the absence of a protein or transcript found in the normal
organ-specific panel may also be an indicator of a perturbed
state.
[0520] In an additional embodiment, the present invention can be
used to determine distinct normal organ-specific blood
fingerprints, such as in different populations of people. In this
regard, distinct normal patterns of organ-specific blood
fingerprints may have differences in populations of patients that
permit one to stratify patients into classes that would respond to
a particular therapeutic regimen and those which would not.
[0521] In a further embodiment, the present invention can be used
to determine the risk of developing a particular biological
condition. A statistically significant alteration (e.g., increase
or decrease) in the levels of one or more members of a particular
blood fingerprint may signify a risk of developing a particular
disease, such as a cancer, an autoimmune disease, or other
biological condition.
[0522] To monitor the progression of a disease, or monitor
responses to therapy, one or more organ-specific blood fingerprints
are detected/measured as described herein using any of the methods
as described herein at one time point and detected/measured again
at subsequent time points, thereby monitoring disease progression
or responses to therapy.
[0523] The normal organ-specific blood fingerprints of the present
invention can be used as a baseline for detecting any of a variety
of diseases (or the lack thereof). In certain embodiments, the
organ-specific blood fingerprints of the present invention can be
used to detect cancer. As such, the present invention can be used
to detect, monitor progression of, or monitor therapeutic regimens
for any cancer, including brain cancer, melanoma, non-Hodgkin's
lymphoma, Hodgkin's disease, leukemias, plasmocytomas, sarcomas,
adenomas, gliomas, thymomas, breast cancer, prostate cancer,
colo-rectal cancer, kidney cancer, renal cell carcinoma, uterine
cancer, pancreatic cancer, esophageal cancer, brain cancer, lung
cancer, ovarian cancer, cervical cancer, testicular cancer, gastric
cancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia
(ALL), acute myelogenous leukemia (AML), chronic myelogenous
leukemia (CML), and chronic lymphocytic leukemia (CLL), or other
cancers. In addition, for the white blood cell cancers, cell
sorting can optionally take place so that only analysis of white
blood cells is carried out and thus direct analysis of the
organ-specific proteins or transcripts from the cells that have
been specifically sorted will be accomplished. Moreover, it should
be understood that any condition, such as a chronic disease, to
cancer to infectious diseases may change the blood immune cells in
specific ways that will be revealed by organ-specific (or cell-type
specific) analyses.
[0524] In certain embodiments, the organ-specific blood
fingerprints of the present invention can be used to detect, to
monitor progression of, or monitor therapeutic regimens for
diseases of the heart, kidney, ureter, bladder, urethra, liver,
prostate, heart, blood vessels, bone marrow, skeletal muscle,
smooth muscle, various specific regions of the brain (including,
but not limited to the amygdala, caudate nucleus, cerebellum,
corpus callosum, fetal, hypothalamus, thalamus), spinal cord,
peripheral nerves, retina, nose, trachea, lungs, mouth, salivary
gland, esophagus, stomach, small intestines, large intestines,
hypothalamus, pituitary, thyroid, pancreas, adrenal glands,
ovaries, oviducts, uterus, placenta, vagina, mammary glands,
testes, seminal vesicles, penis, lymph nodes, thymus, and spleen.
The present invention can be used to detect, to monitor progression
of, or monitor therapeutic regimens for cardiovascular diseases,
neurological diseases, metabolic diseases, respiratory diseases,
autoimmune disease and lung diseases. As would be recognized by the
skilled artisan, the present invention can be used to detect,
monitor the progression of, or monitor treatment for, virtually any
disease wherein the disease causes perturbation in organ-specific
proteins.
[0525] In certain embodiments, the organ-specific blood
fingerprints of the present invention can be used to detect
autoimmune disease. As such, the present invention can be used to
detect, monitor progression of, or monitor therapeutic regimens for
autoimmune diseases such as, but not limited to, rheumatoid
arthritis, multiple sclerosis, insulin dependent diabetes, Addisons
disease, celiac disease, chronic fatigue syndrome, inflammatory
bowel disease, ulcerative colitis, Crohn's disease, Fibromyalgia,
systemic lupus erythematosus, psoriasis, Sjogren's syndrome,
hyperthyroidism/Graves disease, hypothyroidism/Hashimoto's disease,
Insulin-dependent diabetes (type 1), Myasthenia Gravis,
endometriosis, scleroderma, pernicious anemia, Goodpasture
syndrome, Wegener's disease, glomerulonephritis, aplastic anemia,
paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome,
idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia,
Evans syndrome, Factor VIII inhibitor syndrome, systemic
vasculitis, dermatomyositis, polymyositis and rheumatic fever.
[0526] In certain embodiments, the organ-specific blood
fingerprints of the present invention can be used to detect
diseases associated with infections with any of a variety of
infectious organisms, such as viruses, bacteria, parasites and
fungi. Infectious organisms may comprise viruses, (e.g., RNA
viruses, DNA viruses, human immunodeficiency virus (HIV), hepatitis
A, B, and C virus, herpes simplex virus (HSV), cytomegalovirus
(CMV) Epstein-Barr virus (EBV), human papilloma virus (HPV)),
parasites (e.g., protozoan and metazoan pathogens such as Plasmodia
species, Leishmania species, Schistosoma species, Trypanosoma
species), bacteria (e.g., Mycobacteria, in particular, M.
tuberculosis, Salmonella, Streptococci, E. coli, Staphylococci),
fungi (e.g., Candida species, Aspergillus species), Pneumocystis
carinii, and prions.
[0527] The diagnostic panels and generally, methods used for
detecting normal serum organ-specific proteins, can be used to
define/identify disease-associated organ-specific blood
fingerprints. A disease-associated organ-specific blood fingerprint
is a data set comprising the determined level in a blood sample
from an individual afflicted with a disease of one or more
components of a normal serum organ-specific protein set that
demonstrates a statistically significant change as compared to the
determined normal level (e.g., wherein the level in the disease
sample is above or below a predetermined normal range). The data
set is compiled from samples from individuals who are determined to
have a particular disease using established medical diagnostics for
the particular disease. The determined blood (serum) level of each
protein member of a normal serum organ-specific protein set as
measured in the blood of the diseased sample is compared to the
corresponding predetermined normal level. A statistically
significant variation from the predetermined normal level for one
or more members of the normal serum organ-specific protein set
provides diagnostically useful information (disease-associated
fingerprint) for that disease. Note that it may be determined for a
particular disease or disease state that the level of only a few
members of the normal serum organ-specific protein set change
relative to the normal levels. Thus, a disease-associated
organ-specific blood fingerprint may comprise the determined levels
in the blood of only a subset of the components of a normal serum
organ-specific protein set for a given organ and a particular
disease. Thus, a disease-associated organ-specific blood
fingerprint comprises the determined levels in blood of N members
of a serum organ-specific protein set wherein N is 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110 or
any integer value therebetween or more members up to the total
number of members in a given serum organ-specific protein set. In
this regard, in certain embodiments, a disease-associated
organ-specific blood fingerprint comprises the determined levels of
one or more components of a normal serum organ-specific protein
set. In one embodiment, a disease-associated organ-specific blood
fingerprint comprises the determined levels in a sample from an
individual known to have a particular disease of at least two
components of a normal serum organ-specific protein set. In other
embodiments, a disease-associated organ-specific blood fingerprint
comprises the determined levels in a sample from an individual
known to have a particular disease of at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110 or any
integer value therebetween components of a normal serum
organ-specific protein set.
[0528] In certain embodiments, a disease-associated organ-specific
blood fingerprint comprises the determined level in the blood of
components from multiple organs. As noted elsewhere, in certain
embodiments, a disease can impact multiple organs with the result
being a change in blood level of proteins from more than one
organ-specific protein set. Therefore, in certain embodiments, a
disease-associated organ-specific fingerprint comprises the
determined level in the blood of components from 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110 or
any integer value therebetween or more organ-specific protein
sets.
[0529] It should be noted that, in certain embodiments, a
disease-associated organ-specific fingerprint will comprise the
determined level of one or more components of a normal
organ-specific protein set that are NOT components of the
corresponding normal organ-specific protein set. Thus, in this
regard, a disease-associated organ-specific blood fingerprint may
comprise the determined level of one or more components of a normal
organ-specific protein set. Further, in certain embodiments, a
disease-associated "organ-specific" blood fingerprint comprises the
determined levels of one or more components of 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110 or any
integer value therebetween or more normal serum organ-specific
protein sets. Thus, in this regard, a disease-associated
organ-specific blood fingerprint may comprise the determined levels
of one or more components from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110 or any integer value
therebetween or more normal serum organ-specific protein sets.
[0530] One of ordinary skill in the art could readily conclude that
the present invention is useful in defining the normal parameters
for any number of organs in the body. To that end, the present
invention may also be used to define subclinical perturbations from
normal during annual screenings that could be utilized to initiate
therapy or more aggressive examinations at an earlier date.
Further, defining normal for two, three, or more related organs can
be accomplished by the present invention. Such groupings would be
clear to those of skill in the art and could be any of a variety,
include those related to cardiovascular health, including the
heart, lungs, liver, etc. As well as looking at groupings of liver
and blood for infectious and parasitic diseases such as malaria,
HIV, etc.
[0531] Using the diagnostic panels and methods described herein, a
vast array of disease-associated organ-specific blood fingerprints
can be defined for any of a variety of diseases as described
further herein. As such, the present invention further provides
information databases comprising data that make up blood
fingerprints as described herein. As such, the databases may
comprise the defined differential expression levels as determined
using any of a variety of methods such as those described herein,
of each of the plurality of organ-specific proteins that make up a
given fingerprint in any of a variety of settings (e.g., normal or
disease fingerprints).
Targeting for Treatment or Imaging
[0532] In the present specification, the invention describes the
identification of various polypeptides (and their encoding nucleic
acids or fragments thereof) which are expressed as organ-specific
transcripts and in particular embodiments secreted organ-specific
proteins as compared to other organs.
[0533] Accordingly, in one embodiment of the present invention, the
invention provides an isolated nucleic acid molecule having a
nucleotide sequence that encodes an organ-specific target
polypeptide or fragment thereof.
[0534] In certain aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA
molecule encoding a full-length organ-specific polypeptide having
an amino acid sequence as disclosed herein, an organ-specific
polypeptide amino acid sequence lacking the signal peptide as
disclosed herein, an extracellular domain of a transmembrane
organ-specific polypeptide, with or without the signal peptide, as
disclosed herein or any other specifically defined fragment of a
full-length organ-specific polypeptide amino acid sequence as
disclosed herein, or (b) the complement of the DNA molecule of
(a).
[0535] In other aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA
molecule comprising the coding sequence of a full-length
organ-specific polypeptide cDNA as disclosed herein, the coding
sequence of an organ-specific polypeptide lacking the signal
peptide as disclosed herein, the coding sequence of an
extracellular domain of a transmembrane organ-specific polypeptide,
with or without the signal peptide, as disclosed herein or the
coding sequence of any other specifically defined fragment of the
full-length organ-specific polypeptide amino acid sequence as
disclosed herein, or (b) the complement of the DNA molecule of
(a).
[0536] In further aspects, the invention concerns an isolated
nucleic acid molecule comprising a nucleotide sequence having at
least about 80% nucleic acid sequence identity, alternatively at
least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid
sequence identity, to (a) a DNA molecule that encodes the same
mature polypeptide encoded by the full-length coding region of any
of the human protein cDNAs as disclosed herein, or (b) the
complement of the DNA molecule of (a).
[0537] In other aspects, the present invention is directed to
isolated nucleic acid molecules which hybridize to (a) a nucleotide
sequence encoding an organ-specific polypeptide having a
full-length amino acid sequence as disclosed herein or any other
specifically defined fragment of a full-length organ-specific
polypeptide amino acid sequence as disclosed herein, or (b) the
complement of the nucleotide sequence of (a). In this regard, an
embodiment of the present invention is directed to fragments of a
full-length organ-specific polypeptide coding sequence, or the
complement thereof, as disclosed herein, that may find use as, for
example, hybridization probes useful as, for example, diagnostic
probes, antisense oligonucleotide probes, or for encoding fragments
of a full-length organ-specific polypeptide that may optionally
encode a polypeptide comprising a binding site for an
anti-organ-specific polypeptide antibody, an organ-specific binding
oligopeptide or other small organic molecule that binds to an
organ-specific polypeptide. Such nucleic acid fragments are usually
at least about 5 nucleotides in length, alternatively at least
about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,
145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210,
220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340,
350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,
480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,
610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730,
740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,
870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or
1000 nucleotides in length, wherein in this context the term
"about" means the referenced nucleotide sequence length plus or
minus 10% of that referenced length. It is noted that novel
fragments of an organ-specific polypeptide-encoding nucleotide
sequence may be determined in a routine manner by aligning the
organ-specific polypeptide-encoding nucleotide sequence with other
known nucleotide sequences using any of a number of well known
sequence alignment programs and determining which organ-specific
polypeptide-encoding nucleotide sequence fragment(s) are novel. All
of such novel fragments of organ-specific polypeptide-encoding
nucleotide sequences are contemplated herein. Also contemplated are
the organ-specific polypeptide fragments encoded by these
nucleotide molecule fragments, preferably those organ-specific
polypeptide fragments that comprise a binding site for an
anti-organ-specific antibody, an organ-specific binding
oligopeptide or other small organic molecule that binds to an
organ-specific polypeptide.
[0538] In another embodiment, the invention provides isolated
organ-specific polypeptides encoded by any of the isolated nucleic
acid sequences hereinabove identified.
[0539] In another embodiment, the invention provides an antibody
which binds, preferably specifically, to any of the above or below
described polypeptides. Optionally, the antibody is a monoclonal
antibody, antibody fragment, chimeric antibody, humanized antibody,
single-chain antibody or antibody that competitively inhibits the
binding of an anti-organ-specific polypeptide antibody to its
respective antigenic epitope. Antibodies of the present invention
may optionally be conjugated to a growth inhibitory agent or
cytotoxic agent such as a toxin, including, for example, a
maytansinoid or calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. The antibodies of the
present invention may optionally be produced in CHO cells or
bacterial cells and preferably induce death of a cell to which they
bind. For diagnostic purposes, the antibodies of the present
invention may be detectably labeled, attached to a solid support,
or the like.
[0540] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described antibodies. Host cell comprising any such vector are also
provided. By way of example, the host cells may be CHO cells, E.
coli cells, or yeast cells. A process for producing any of the
herein described antibodies is further provided and comprises
culturing host cells under conditions suitable for expression of
the desired antibody and recovering the desired antibody from the
cell culture.
[0541] In another embodiment, the invention provides oligopeptides
("organ-specific binding oligopeptides") which bind, preferably
specifically, to any of the above or below described organ-specific
polypeptides. Optionally, the organ-specific binding oligopeptides
of the present invention may be conjugated to a growth inhibitory
agent or cytotoxic agent such as a toxin, including, for example, a
maytansinoid or calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. The organ-specific
binding oligopeptides of the present invention may optionally be
produced in CHO cells or bacterial cells and preferably induce
death of a cell to which they bind. For diagnostic purposes, the
organ-specific binding oligopeptides of the present invention may
be detectably labeled, attached to a solid support, or the
like.
[0542] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described organ-specific binding oligopeptides. Host cell
comprising any such vector are also provided. By way of example,
the host cells may be CHO cells, E. coli cells, or yeast cells. A
process for producing any of the herein described organ-specific
binding oligopeptides is further provided and comprises culturing
host cells under conditions suitable for expression of the desired
oligopeptide and recovering the desired oligopeptide from the cell
culture.
[0543] In another embodiment, the invention provides small organic
molecules ("organ-specific binding organic molecules") which bind,
preferably specifically, to any of the above or below described
organ-specific polypeptides. Optionally, the organ-specific binding
organic molecules of the present invention may be conjugated to a
growth inhibitory agent or cytotoxic agent such as a toxin,
including, for example, a maytansinoid or calicheamicin, an
antibiotic, a radioactive isotope, a nucleolytic enzyme, or the
like. The organ-specific binding organic molecules of the present
invention preferably induce death of a cell to which they bind. For
diagnostic purposes, the organ-specific binding organic molecules
of the present invention may be detectably labeled, attached to a
solid support, or the like.
[0544] In a still further embodiment, the invention concerns a
composition of matter comprising an organ-specific polypeptide as
described herein, a chimeric organ-specific polypeptide as
described herein, an anti-organ-specific antibody as described
herein, an organ-specific binding oligopeptide as described herein,
or an organ-specific binding organic molecule as described herein,
in combination with a carrier. Optionally, the carrier is a
pharmaceutically acceptable carrier.
[0545] In yet another embodiment, the invention concerns an article
of manufacture comprising a container and a composition of matter
contained within the container, wherein the composition of matter
may comprise an organ-specific polypeptide as described herein, a
chimeric organ-specific polypeptide as described herein, an
anti-organ-specific antibody as described herein, an organ-specific
binding oligopeptide as described herein, or an organ-specific
binding organic molecule as described herein. The article may
further optionally comprise a label affixed to the container, or a
package insert included with the container, that refers to the use
of the composition of matter for the therapeutic treatment or
diagnostic detection of a tumor.
[0546] Another embodiment of the present invention is directed to
the use of an organ-specific polypeptide as described herein, a
chimeric organ-specific polypeptide as described herein, an
anti-organ-specific polypeptide antibody as described herein, an
organ-specific binding oligopeptide as described herein, or an
organ-specific binding organic molecule as described herein, for
the preparation of a medicament useful in the treatment of a
condition which is responsive to the organ-specific polypeptide,
chimeric organ-specific polypeptide, anti-organ-specific
polypeptide antibody, organ-specific binding oligopeptide, or
organ-specific binding organic molecule.
[0547] Another embodiment of the present invention is directed to a
method for inhibiting the growth of a cell that expresses an
organ-specific polypeptide, wherein the method comprises contacting
the cell with an antibody, an oligopeptide or a small organic
molecule that binds to the organ-specific polypeptide, and wherein
the binding of the antibody, oligopeptide or organic molecule to
the organ-specific polypeptide causes inhibition of the growth of
the cell expressing the organ-specific polypeptide. In preferred
embodiments, the cell is a cancer cell or disease harboring cell
and binding of the antibody, oligopeptide or organic molecule to
the organ-specific polypeptide causes death of the cell expressing
the organ-specific polypeptide. Optionally, the antibody is a
monoclonal antibody, antibody fragment, chimeric antibody,
humanized antibody, or single-chain antibody. Antibodies,
organ-specific binding oligopeptides and organ-specific binding
organic molecules employed in the methods of the present invention
may optionally be conjugated to a growth inhibitory agent or
cytotoxic agent such as a toxin, including, for example, a
maytansinoid or calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. The antibodies and
organ-specific binding oligopeptides employed in the methods of the
present invention may optionally be produced in CHO cells or
bacterial cells.
[0548] Yet another embodiment of the present invention is directed
to a method of therapeutically treating a mammal having a cancerous
cells or disease containing cells or tissues comprising cells that
express an organ-specific polypeptide, wherein the method comprises
administering to the mammal a therapeutically effective amount of
an antibody, an oligopeptide or a small organic molecule that binds
to the organ-specific polypeptide, thereby resulting in the
effective therapeutic treatment of the tumor. Optionally, the
antibody is a monoclonal antibody, antibody fragment, chimeric
antibody, humanized antibody, or single-chain antibody. Antibodies,
organ-specific binding oligopeptides and organ-specific binding
organic molecules employed in the methods of the present invention
may optionally be conjugated to a growth inhibitory agent or
cytotoxic agent such as a toxin, including, for example, a
maytansinoid or calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. The antibodies and
oligopeptides employed in the methods of the present invention may
optionally be produced in CHO cells or bacterial cells.
[0549] Yet another embodiment of the present invention is directed
to a method of determining the presence of an organ-specific
polypeptide in a sample suspected of containing the organ-specific
polypeptide, wherein the method comprises exposing the sample to an
antibody, oligopeptide or small organic molecule that binds to the
organ-specific polypeptide and determining binding of the antibody,
oligopeptide or organic molecule to the organ-specific polypeptide
in the sample, wherein the presence of such binding is indicative
of the presence of the organ-specific polypeptide in the sample.
Optionally, the sample may contain cells (which may be cancer
cells) suspected of expressing the organ-specific polypeptide. The
antibody, organ-specific binding oligopeptide or organ-specific
binding organic molecule employed in the method may optionally be
detectably labeled, attached to a solid support, or the like.
[0550] A further embodiment of the present invention is directed to
a method of diagnosing the presence of a tumor in a mammal, wherein
the method comprises detecting the level of expression of a gene
encoding an organ-specific polypeptide (a) in a test sample of
tissue cells obtained from said mammal, and (b) in a control sample
of known normal non-cancerous cells of the same tissue origin or
type, wherein a higher level of expression of the organ-specific
polypeptide in the test sample, as compared to the control sample,
is indicative of the presence of tumor in the mammal from which the
test sample was obtained.
[0551] Another embodiment of the present invention is directed to a
method of diagnosing the presence of a tumor in a mammal, wherein
the method comprises (a) contacting a test sample comprising tissue
cells obtained from the mammal with an antibody, oligopeptide or
small organic molecule that binds to an organ-specific polypeptide
and (b) detecting the formation of a complex between the antibody,
oligopeptide or small organic molecule and the organ-specific
polypeptide in the test sample, wherein the formation of a complex
is indicative of the presence of a tumor in the mammal. Optionally,
the antibody, organ-specific binding oligopeptide or organ-specific
binding organic molecule employed is detectably labeled, attached
to a solid support, or the like, and/or the test sample of tissue
cells is obtained from an individual suspected of having a
cancerous tumor.
[0552] Yet another embodiment of the present invention is directed
to a method for treating or preventing a cell proliferative
disorder associated with altered, preferably increased, expression
or activity of an organ-specific polypeptide, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of an organ-specific polypeptide.
Preferably, the cell proliferative disorder is cancer and the
antagonist of the organ-specific polypeptide is an
anti-organ-specific polypeptide antibody, organ-specific binding
oligopeptide, organ-specific binding organic molecule or antisense
oligonucleotide. Effective treatment or prevention of the cell
proliferative disorder may be a result of direct killing or growth
inhibition of cells that express an organ-specific polypeptide or
by antagonizing the cell growth potentiating activity of an
organ-specific polypeptide.
[0553] Yet another embodiment of the present invention is directed
to a method of binding an antibody, oligopeptide or small organic
molecule to a cell that expresses an organ-specific polypeptide,
wherein the method comprises contacting a cell that expresses an
organ-specific polypeptide with said antibody, oligopeptide or
small organic molecule under conditions which are suitable for
binding of the antibody, oligopeptide or small organic molecule to
said organ-specific polypeptide and allowing binding
therebetween.
[0554] Other embodiments of the present invention are directed to
the use of (a) an organ-specific polypeptide, (b) a nucleic acid
encoding an organ-specific polypeptide or a vector or host cell
comprising that nucleic acid, (c) an anti-organ-specific
polypeptide antibody, (d) an organ-specific-binding oligopeptide,
or (e) an organ-specific-binding small organic molecule in the
preparation of a medicament useful for (i) the therapeutic
treatment or diagnostic detection of a cancer or tumor, or (ii) the
therapeutic treatment or prevention of a cell proliferative
disorder.
[0555] Another embodiment of the present invention is directed to a
method for inhibiting the growth of a cancer cell, wherein the
growth of said cancer cell is at least in part dependent upon the
growth potentiating effect(s) of an organ-specific polypeptide
(wherein the organ-specific polypeptide may be expressed either by
the cancer cell itself or a cell that produces polypeptide(s) that
have a growth potentiating effect on cancer cells), wherein the
method comprises contacting the organ-specific polypeptide with an
antibody, an oligopeptide or a small organic molecule that binds to
the organ-specific polypeptide, thereby antagonizing the
growth-potentiating activity of the organ-specific polypeptide and,
in turn, inhibiting the growth of the cancer cell. Preferably the
growth of the cancer cell is completely inhibited. Even more
preferably, binding of the antibody, oligopeptide or small organic
molecule to the organ-specific polypeptide induces the death of the
cancer cell. Optionally, the antibody is a monoclonal antibody,
antibody fragment, chimeric antibody, humanized antibody, or
single-chain antibody. Antibodies, organ-specific binding
oligopeptides and organ-specific binding organic molecules employed
in the methods of the present invention may optionally be
conjugated to a growth inhibitory agent or cytotoxic agent such as
a toxin, including, for example, a maytansinoid or calicheamicin,
an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the
like. The antibodies and organ-specific binding oligopeptides
employed in the methods of the present invention may optionally be
produced in CHO cells or bacterial cells.
[0556] Yet another embodiment of the present invention is directed
to a method of therapeutically treating a tumor in a mammal,
wherein the growth of said tumor is at least in part dependent upon
the growth potentiating effect(s) of an organ-specific polypeptide,
wherein the method comprises administering to the mammal a
therapeutically effective amount of an antibody, an oligopeptide or
a small organic molecule that binds to the organ-specific
polypeptide, thereby antagonizing the growth potentiating activity
of said organ-specific polypeptide and resulting in the effective
therapeutic treatment of the tumor. Optionally, the antibody is a
monoclonal antibody, antibody fragment, chimeric antibody,
humanized antibody, or single-chain antibody. Antibodies,
organ-specific binding oligopeptides and organ-specific binding
organic molecules employed in the methods of the present invention
may optionally be conjugated to a growth inhibitory agent or
cytotoxic agent such as a toxin, including, for example, a
maytansinoid or calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. The antibodies and
oligopeptides employed in the methods of the present invention may
optionally be produced in CHO cells or bacterial cells.
Anti-Organ-Specific Polypeptide Antibodies
[0557] In one embodiment, the present invention provides
anti-organ-specific antibodies which may find use herein as
therapeutic, diagnostic, and/or imaging agents. Exemplary
antibodies include polyclonal, monoclonal, humanized, bispecific,
and heteroconjugate antibodies.
[0558] 1. Polyclonal Antibodies
[0559] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen (especially when synthetic peptides are used)
to a protein that is immunogenic in the species to be immunized.
For example, the antigen can be conjugated to keyhole limpet
hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor, using a bifunctional or derivatizing agent,
e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through
cysteine residues), N-hydroxysuccinimide (through lysine residues),
glutaraldehyde, succinic anhydride, SOCl.sub.2, or
R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are different alkyl
groups.
[0560] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later, the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later, the animals
are bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are suitably used to enhance the immune
response.
[0561] 2. Monoclonal Antibodies
[0562] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0563] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as described above to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
After immunization, lymphocytes are isolated and then fused with a
myeloma cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press,
1986)).
[0564] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium which medium preferably contains one or
more substances that inhibit the growth or survival of the unfused,
parental myeloma cells (also referred to as fusion partner). For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the selective
culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0565] Preferred fusion partner myelomacells are those that fuse
efficiently, support stable high-level production of antibody by
the selected antibody-producing cells, and are sensitive to a
selective medium that selects against the unfused parental cells.
Preferred myeloma cell lines are murine myeloma lines, such as
those derived from MOPC-21 and MPC-11 mouse tumors available from
the Salk Institute Cell Distribution Center, San Diego, Calif. USA,
and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the
American Type Culture Collection, Manassas, Va., USA. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987)).
[0566] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA).
[0567] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis described in
Munson et al., Anal. Biochem., 107:220 (1980).
[0568] Once hybridoma cells that produce antibodies of the desired
specificity, affinity, and/or activity are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal e.g., by i.p. injection of the cells
into mice.
[0569] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, affinity chromatography (e.g., using protein A or protein
G-Sepharose) or ion-exchange chromatography, hydroxylapatite
chromatography, gel electrophoresis, dialysis, etc.
[0570] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol.,
5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188
(1992).
[0571] In a further embodiment, monoclonal antibodies or antibody
fragments can be isolated from antibody phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and
Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0572] The DNA that encodes the antibody may be modified to produce
chimeric or fusion antibody polypeptides, for example, by
substituting human heavy chain and light chain constant domain
(C.sub.H and C.sub.L) sequences for the homologous murine sequences
(U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl. Acad.
Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding
sequence with all or part of the coding sequence for a
non-immunoglobulin polypeptide (heterologous polypeptide). The
non-immunoglobulin polypeptide sequences can substitute for the
constant domains of an antibody, or they are substituted for the
variable domains of one antigen-combining site of an antibody to
create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0573] 3. Human and Humanized Antibodies
[0574] The anti-organ-specific antibodies of the invention may
further comprise humanized antibodies or human antibodies.
Humanized forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0575] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al. Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0576] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity and HAMA response (human anti-mouse antibody)
when the antibody is intended for human therapeutic use. According
to the so-called "best-fit" method, the sequence of the variable
domain of a rodent antibody is screened against the entire library
of known human variable domain sequences. The human V domain
sequence which is closest to that of the rodent is identified and
the human framework region (FR) within it accepted for the
humanized antibody (Sims et al., J. Immunol. 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)).
[0577] Another method uses a particular framework region derived
from the consensus sequence of all human antibodies of a particular
subgroup of light or heavy chains. The same framework may be used
for several different humanized antibodies (Carter et al., Proc.
Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol.
151:2623 (1993)).
[0578] It is further important that antibodies be humanized with
retention of high binding affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
a preferred method, humanized antibodies are prepared by a process
of analysis of the parental sequences and various conceptual
humanized products using three-dimensional models of the parental
and humanized sequences. Three-dimensional immunoglobulin models
are commonly available and are familiar to those skilled in the
art. Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0579] Various forms of a humanized anti-organ-specific antibody
are contemplated. For example, the humanized antibody may be an
antibody fragment, such as a Fab, which is optionally conjugated
with one or more cytotoxic agent(s) in order to generate an
immunoconjugate. Alternatively, the humanized antibody may be an
intact antibody, such as an intact IgG1 antibody.
[0580] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33
(1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of
GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852.
[0581] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553) can be used to produce human antibodies and
antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell. Phage display can be performed in a variety of formats,
reviewed in, e.g., Johnson, Kevin S, and Chiswell, David J.,
Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of probes (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0582] As discussed above, human antibodies may also be generated
by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and
5,229,275).
[0583] 4. Antibody Fragments
[0584] In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. The smaller size
of the fragments allows for rapid clearance, and may lead to
improved access to solid tumors.
[0585] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising a salvage receptor binding epitope
residues are described in U.S. Pat. No. 5,869,046. Other techniques
for the production of antibody fragments will be apparent to the
skilled practitioner. In other embodiments, the antibody of choice
is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat.
No. 5,571,894; and U.S. Pat. No. 5,587,458. Fv and sFv are the only
species with intact combining sites that are devoid of constant
regions; thus, they are suitable for reduced nonspecific binding
during in vivo use. sFv fusion proteins may be constructed to yield
fusion of an effector protein at either the amino or the carboxy
terminus of an sFv. See Antibody Engineering, ed. Borrebaeck,
supra. The antibody fragment may also be a "linear antibody", e.g.,
as described in U.S. Pat. No. 5,641,870 for example. Such linear
antibody fragments may be monospecific or bispecific.
[0586] 5. Bispecific Antibodies
[0587] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of an
organ-specific protein as described herein. Other such antibodies
may combine an organ-specific binding site with a binding site for
another protein. Alternatively, an anti-organ-specific arm may be
combined with an arm which binds to a triggering molecule on a
leukocyte such as a T-cell receptor molecule (e.g. CD3), or Fc
receptors for IgG (Fc.gamma.R), such as Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16), so as to focus and
localize cellular defense mechanisms to the
organ-specific-expressing cell. Bispecific antibodies may also be
used for diagnostic purposes, attaching imaging agents or
localizing cytotoxic agents to cells which express organ-specific
transcripts and/or polypeptides. These antibodies possess an
organ-specific-binding arm and an arm which binds the cytotoxic
agent (e.g., saporin, anti-interferon-.alpha., vinca alkaloid,
ricin A chain, methotrexate or radioactive isotope hapten).
Bispecific antibodies can be prepared as full length antibodies or
antibody fragments (e.g., F(ab').sub.2 bispecific antibodies).
[0588] WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc .alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0589] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J. 10:3655-3659
(1991).
[0590] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. It is preferred to have the first heavy-chain constant
region (C.sub.H1) containing the site necessary for light chain
bonding, present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host cell. This
provides for greater flexibility in adjusting the mutual
proportions of the three polypeptide fragments in embodiments when
unequal ratios of the three polypeptide chains used in the
construction provide the optimum yield of the desired bispecific
antibody. It is, however, possible to insert the coding sequences
for two or all three polypeptide chains into a single expression
vector when the expression of at least two polypeptide chains in
equal ratios results in high yields or when the ratios have no
significant affect on the yield of the desired chain
combination.
[0591] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology 121:210 (1986).
[0592] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the C.sub.H3 domain. In this method,
one or more small amino acid side chains from the interface of the
first antibody molecule are replaced with larger side chains (e.g.,
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g., alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0593] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0594] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent, sodium arsenite, to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0595] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets. Various techniques for making and isolating
bispecific antibody fragments directly from recombinant cell
culture have also been described. For example, bispecific
antibodies have been produced using leucine zippers. Kostelny et
al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper
peptides from the Fos and Jun proteins were linked to the Fab'
portions of two different antibodies by gene fusion. The antibody
homodimers were reduced at the hinge region to form monomers and
then re-oxidized to form the antibody heterodimers. This method can
also be utilized for the production of antibody homodimers. The
"diabody" technology described by Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative
mechanism for making bispecific antibody fragments. The fragments
comprise a V.sub.H connected to a V.sub.L by a linker which is too
short to allow pairing between the two domains on the same chain.
Accordingly, the V.sub.H and V.sub.L domains of one fragment are
forced to pair with the complementary V.sub.L and V.sub.H domains
of another fragment, thereby forming two antigen-binding sites.
Another strategy for making bispecific antibody fragments by the
use of single-chain Fv (sFv) dimers has also been reported. See
Gruber et al., J. Immunol., 152:5368 (1994).
[0596] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0597] 6. Heteroconjugate Antibodies
[0598] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells [U.S.
Pat. No. 4,676,980], and for treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0599] 7. Multivalent Antibodies
[0600] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present invention can be multivalent antibodies (which are other
than of the IgM class) with three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody can comprise a
dimerization domain and three or more antigen binding sites. The
preferred dimerization domain comprises (or consists of) an Fc
region or a hinge region. In this scenario, the antibody will
comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fc region. The preferred multivalent antibody
herein comprises (or consists of) three to about eight, but
preferably four, antigen binding sites. The multivalent antibody
comprises at least one polypeptide chain (and preferably two
polypeptide chains), wherein the polypeptide chain(s) comprise two
or more variable domains. For instance, the polypeptide chain(s)
may comprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a
first variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CHI-flexible linker-VH-CHI-Fc region
chain; or VH-CHI-VH-CHI-Fc region chain. The multivalent antibody
herein preferably further comprises at least two (and preferably
four) light chain variable domain polypeptides. The multivalent
antibody herein may, for instance, comprise from about two to about
eight light chain variable domain polypeptides. The light chain
variable domain polypeptides contemplated here comprise a light
chain variable domain and, optionally, further comprise a CL
domain.
[0601] 8. Effector Function Engineering
[0602] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g., so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.,
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design 3:219-230 (1989). To increase the
serum half life of the antibody, one may incorporate a salvage
receptor binding epitope into the antibody (especially an antibody
fragment) as described in U.S. Pat. No. 5,739,277, for example. As
used herein, the term "salvage receptor binding epitope" refers to
an epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0603] 9. Immunoconjugate
[0604] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g.,
an enzymatically active toxin of bacterial, fungal, plant, or
animal origin, or fragments thereof), or a radioactive isotope
(i.e., a radioconjugate).
[0605] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCL), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutareldehyde),
bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0606] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
[0607] 10. Immunoliposomes
[0608] The anti-organ-specific antibodies disclosed herein may also
be formulated as immunoliposomes. A "liposome" is a small vesicle
composed of various types of lipids, phospholipids and/or
surfactant which is useful for delivery of a drug to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0609] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al., J. National Cancer Inst.
81(19):1484 (1989).
[0610] B. Organ-Specific Binding Oligopeptides
[0611] Organ-specific binding oligopeptides of the present
invention are oligopeptides that bind, preferably specifically, to
an organ-specific polypeptide as described herein. organ-specific
binding oligopeptides may be chemically synthesized using known
oligopeptide synthesis methodology or may be prepared and purified
using recombinant technology. organ-specific binding oligopeptides
are usually at least about 5 amino acids in length, alternatively
at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids
in length or more, wherein such oligopeptides that are capable of
binding, preferably specifically, to an organ-specific polypeptide
as described herein. organ-specific binding oligopeptides may be
identified without undue experimentation using well known
techniques. In this regard, it is noted that techniques for
screening oligopeptide libraries for oligopeptides that are capable
of specifically binding to a polypeptide target are well known in
the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871,
4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT
Publication Nos. WO 84/03506 and WO084/03564; Geysen et al., Proc.
Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc.
Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in
Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J.
Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,
140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad.
Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry,
30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D.
et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991)
Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)
Current Opin. Biotechnol., 2:668).
[0612] In this regard, bacteriophage (phage) display is one well
known technique which allows one to screen large oligopeptide
libraries to identify member(s) of those libraries which are
capable of specifically binding to a polypeptide target. Phage
display is a technique by which variant polypeptides are displayed
as fusion proteins to the coat protein on the surface of
bacteriophage particles (Scott, J. K. and Smith, G. P. (1990)
Science 249: 386). The utility of phage display lies in the fact
that large libraries of selectively randomized protein variants (or
randomly cloned cDNAs) can be rapidly and efficiently sorted for
those sequences that bind to a target molecule with high affinity.
Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad.
Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991)
Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352:
624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A.
S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on
phage have been used for screening millions of polypeptides or
oligopeptides for ones with specific binding properties (Smith, G.
P. (1991) Current Opin. Biotechnol., 2:668). Sorting phage
libraries of random mutants requires a strategy for constructing
and propagating a large number of variants, a procedure for
affinity purification using the target receptor, and a means of
evaluating the results of binding enrichments. U.S. Pat. Nos.
5,223,409, 5,403,484, 5,571,689, and 5,663,143.
[0613] Although most phage display methods have used filamentous
phage, lambdoid phage display systems (WO95/34683; U.S. Pat. No.
5,627,024), T4 phagedisplay systems (Ren, Z-J. et al. (1998) Gene
215:439; Zhu, Z. (1997) CAN 33:534; Jiang, J. et al. (1997) can
128:44380; Ren, Z-J. et al. (1997) CAN 127:215644; Ren, Z-J. (1996)
Protein Sci. 5:1833; Efimov, V. P. et al. (1995) Virus Genes
10:173) and T7 phage display systems (Smith, G. P. and Scott, J. K.
(1993) Methods in Enzymology, 217, 228-257; U.S. Pat. No.
5,766,905) are also known.
[0614] Many other improvements and variations of the basic phage
display concept have now been developed. These improvements enhance
the ability of display systems to screen peptide libraries for
binding to selected target molecules and to display functional
proteins with the potential of screening these proteins for desired
properties. Combinatorial reaction devices for phage display
reactions have been developed (WO 98/14277) and phage display
libraries have been used to analyze and control bimolecular
interactions (WO 98/20169; WO 98/20159) and properties of
constrained helical peptides (WO 98/20036). WO 97/35196 describes a
method of isolating an affinity ligand in which a phage display
library is contacted with one solution in which the ligand will
bind to a target molecule and a second solution in which the
affinity ligand will not bind to the target molecule, to
selectively isolate binding ligands. WO 97/46251 describes a method
of biopanning a random phage display library with an affinity
purified antibody and then isolating binding phage, followed by a
micropanning process using microplate wells to isolate high
affinity binding phage. The use of Staphlylococcus aureus protein A
as an affinity tag has also been reported (Li et al. (1998) Mol.
Biotech., 9:187). WO 97/47314 describes the use of substrate
subtraction libraries to distinguish enzyme specificities using a
combinatorial library which may be a phage display library. A
method for selecting enzymes suitable for use in detergents using
phage display is described in WO 97/09446. Additional methods of
selecting specific binding proteins are described in U.S. Pat. Nos.
5,498,538, 5,432,018, and WO 98/15833.
[0615] Methods of generating peptide libraries and screening these
libraries are also disclosed in U.S. Pat. Nos. 5,723,286,
5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018,
5,698,426, 5,763,192, and 5,723,323.
[0616] C. Organ-Specific Binding Organic Molecules
[0617] Organ-specific binding organic molecules are organic
molecules other than oligopeptides or antibodies as defined herein
that bind, preferably specifically, to an organ-specific
polypeptide as described herein. organ-specific binding organic
molecules may be identified and chemically synthesized using known
methodology (see, e.g., PCT Publication Nos. WO00/00823 and
WO00/39585). organ-specific binding organic molecules are usually
less than about 2000 daltons in size, alternatively less than about
1500, 750, 500, 250 or 200 daltons in size, wherein such organic
molecules that are capable of binding, preferably specifically, to
an organ-specific polypeptide as described herein may be identified
without undue experimentation using well known techniques. In this
regard, it is noted that techniques for screening organic molecule
libraries for molecules that are capable of binding to a
polypeptide target are well known in the art (see, e.g., PCT
Publication Nos. WO00/00823 and WO00/39585). organ-specific binding
organic molecules may be, for example, aldehydes, ketones, oximes,
hydrazones, semicarbazones, carbazides, primary amines, secondary
amines, tertiary amines, N-substituted hydrazines, hydrazides,
alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids,
esters, amides, ureas, carbamates, carbonates, ketals, thioketals,
acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides,
alkyl sulfonates, aromatic compounds, heterocyclic compounds,
anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines,
oxazolines, thiazolidines, thiazolines, enamines, sulfonamides,
epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo
compounds, acid chlorides, or the like.
[0618] D. Screening for Anti-Organ-Specific Antibodies,
Organ-Specific Binding Oligopeptides and Organ-Specific Binding
Organic Molecules with the Desired Properties
[0619] Techniques for generating antibodies, oligopeptides and
organic molecules that bind to organ-specific polypeptides have
been described above. One may further select antibodies,
oligopeptides or other organic molecules with certain biological
characteristics, as desired.
[0620] The growth inhibitory effects of an anti-organ-specific
antibody, oligopeptide or other organic molecule of the invention
may be assessed by methods known in the art, e.g., using cells
which express an organ-specific polypeptide either endogenously or
following transfection with the organ-specific gene. For example,
appropriate tumor cell lines and organ-specific-transfected cells
may treated with an anti-organ-specific monoclonal antibody,
oligopeptide or other organic molecule of the invention at various
concentrations for a few days (e.g., 2-7) days and stained with
crystal violet or MTT or analyzed by some other colorimetric assay.
Another method of measuring proliferation would be by comparing
.sup.3H-thymidine uptake by the cells treated in the presence or
absence an anti-organ-specific antibody, organ-specific binding
oligopeptide or organ-specific binding organic molecule of the
invention. After treatment, the cells are harvested and the amount
of radioactivity incorporated into the DNA quantitated in a
scintillation counter. Appropriate positive controls include
treatment of a selected cell line with a growth inhibitory antibody
known to inhibit growth of that cell line. Growth inhibition of
tumor cells in vivo can be determined in various ways known in the
art. Preferably, the tumor cell is one that overexpresses an
organ-specific polypeptide. Preferably, the anti-organ-specific
antibody, organ-specific binding oligopeptide or organ-specific
binding organic molecule will inhibit cell proliferation of an
organ-specific-expressing tumor cell in vitro or in vivo by about
25-100% compared to the untreated tumor cell, more preferably, by
about 30-100%, and even more preferably by about 50-100% or
70-100%, in one embodiment, at an antibody concentration of about
0.5 to 30 .mu.g/ml. Growth inhibition can be measured at an
antibody concentration of about 0.5 to 30 .mu.g/ml or about 0.5 nM
to 200 nM in cell culture, where the growth inhibition is
determined 1-10 days after exposure of the tumor cells to the
antibody. The antibody is growth inhibitory in vivo if
administration of the anti-organ-specific antibody at about 1
.mu.g/kg to about 100 mg/kg body weight results in reduction in
tumor size or reduction of tumor cell proliferation within about 5
days to 3 months from the first administration of the antibody,
preferably within about 5 to 30 days.
[0621] To select for an anti-organ-specific antibody,
organ-specific binding oligopeptide or organ-specific binding
organic molecule which induces cell death, loss of membrane
integrity as indicated by, e.g., propidium iodide (PI), trypan blue
or 7AAD uptake may be assessed relative to control. API uptake
assay can be performed in the absence of complement and immune
effector cells. organ-specific polypeptide-expressing tumor cells
are incubated with medium alone or medium containing the
appropriate anti-organ-specific antibody (e.g., at about 10
.mu.g/ml), organ-specific binding oligopeptide or organ-specific
binding organic molecule. The cells are incubated for a 3 day time
period. Following each treatment, cells are washed and aliquoted
into 35 mm strainer-capped 12.times.75 tubes (1 ml per tube, 3
tubes per treatment group) for removal of cell clumps. Tubes then
receive PI (1 .mu.g/ml). Samples may be analyzed using a
FACSCAN.RTM.. flow cytometer and FACSCONVERT.RTM.. CellQuest
software (Becton Dickinson). Those anti-organ-specific antibodies,
organ-specific binding oligopeptides or organ-specific binding
organic molecules that induce statistically significant levels of
cell death as determined by PI uptake may be selected as cell
death-inducing anti-organ-specific antibodies, organ-specific
binding oligopeptides or organ-specific binding organic
molecules.
[0622] To screen for antibodies, oligopeptides or other organic
molecules which bind to an epitope on an organ-specific polypeptide
bound by an antibody of interest, a routine cross-blocking assay
such as that described in Antibodies, A Laboratory Manual, Cold
Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. This assay can be used to determine if a test antibody,
oligopeptide or other organic molecule binds the same site or
epitope as a known anti-organ-specific antibody. Alternatively, or
additionally, epitope mapping can be performed by methods known in
the art. For example, the antibody sequence can be mutagenized such
as by alanine scanning, to identify contact residues. The mutant
antibody is initially tested for binding with polyclonal antibody
to ensure proper folding. In a different method, peptides
corresponding to different regions of an organ-specific polypeptide
can be used in competition assays with the test antibodies or with
a test antibody and an antibody with a characterized or known
epitope.
[0623] Other methods of using the present panels, sets and
individual members of the panels noted herein includes the use for
evaluation of test compounds in a biological system to monitor
changes related thereto. As one of skill in the art could readily
appreciate, when observing a disease related profile, a test
compound that changes said profile to be more similar to the normal
profile is of significant interest as a drug lead. Accordingly, the
present invention also provides a method for optimizing drug/test
compound leads by treating an animal, cell, or tissue with said
compound and observing whether the organ, cell, or tissue-specific
profile changes to deviate from the diseased profile toward a more
normal profile.
[0624] One aspect of the invention is a method for evaluating a
test compound for biological activity, comprising: providing a
database comprising a plurality of reference/normal organ-specific
protein and/or gene expression profiles, each profile comprising a
representation of the expression level of a plurality of genes or
proteins in a test cell exposed to a reference compound and a
representation of the reference compound; providing a test
expression profile, comprising a representation of the expression
level of a plurality of genes and/or proteins in a test cell
exposed to said test compound; comparing said test expression
profile with said first expression profiles; identifying at least
one first expression profile that is similar to said test
expression profile; displaying said selected expression
profile.
[0625] Another aspect of the invention is a system for performing
the method of the invention.
[0626] Another aspect of the invention is a computer-readable
medium having encoded thereon a set of instructions enabling a
computer system to perform the method of the invention.
[0627] An additional aspect of the present invention provides a
system comprising a correlative database that permits one to study
relationships between different genes or polypeptides encoded
thereby, between genes or the polypeptides and a variety of
compounds, to investigate structure-function relationships between
different compounds. The database contains a plurality of standard
gene expression or protein expression profiles of organ-specific
components or under a plurality of specified conditions. The
conditions specified can include expression within a particular
cell type (for example, fibroblast, lymphocyte, neuron, oocyte,
hepatocyte, and the like), expression at a particular point in the
cell cycle (e.g., G1), expression in a specified disease state, the
presence of environmental factors (for example, temperature,
pressure, CO.sub.2 partial pressure, osmotic pressure, shear
stress, confluency, adherence, and the like), the presence of
pathogenic organisms (for example, viruses, bacterial, fungi, and
extra- or intracellular parasites), expression in the presence of
heterologous genes, expression in the presence of test compounds,
and the like, and combinations thereof. The database preferably
comprises expression profiles for at least 10 different genes from
specific to a particular organ or tissue. The standard expression
profiles are preferably annotated, for example, with information
regarding the conditions under which the profile was obtained.
Preferably, the database also contains annotations for one or more
genes, more preferably for each gene represented in the database.
The annotations can include any available information about the
gene, such as, for example, the gene's or protein's names and
synonyms, the sequence, any known biological activity or function,
any genes or proteins of similar sequence, any metabolic or protein
interaction pathways to which it is known to belong, and the
like.
[0628] The database contains interpretive gene expression profiles
and bioassay profiles for a plurality of different compounds that
comprise a representation of a compound's mode of action and/or
toxicity ("drug signatures"), and can include experimental
compounds and/or "standard" compounds. Drug signatures provide a
unique picture of a compound's comprehensive activity in vivo,
including both its effect on gene transcription and its interaction
with proteins. Standard compounds are preferably
well-characterized, and preferably exhibit a known biological
effect on host cells and/or organisms. Standard compounds can
advantageously be selected from the class of available drug
compounds, natural toxins and venoms, known poisons, vitamins and
nutrients, metabolic byproducts, and the like. The standard
compounds can be selected to provide, as a set, a wide range of
different gene expression profiles. The records for the standard
compounds are preferably annotated with information available
regarding the compounds, such as, for example, the compound name,
structure and chemical formula, molecular weight, aqueous
solubility, pH, lipophilicity, known biological activity, source,
proteins and/or genes it is known to interact with, assays for
detecting and/or confirming activity of the compound or related
compounds, and the like. Alternatively, one can employ a database
constructed from random compounds, combinatorial libraries, and the
like.
[0629] The database further contains bioassay data derived from
experiments in which one or more compounds represented in the
database are examined for activity against one or more proteins
represented in the database. Bioassay data can be obtained from
open literature and directly by experiment.
[0630] Further, the database preferably contains product data
related to the compounds, genes, proteins, expression profiles,
and/or bioassay data otherwise present in the database. The product
data can be information regarding physical products, such as
bioassay kits and reagents, compounds useful as positive and
negative controls, compounds similar to the test compound useful
for further study, DNA microarrays and the like, or can comprise
information-based products, such as additional data regarding gene
or protein function and/or relationships (for example, sequence
data from other species, information regarding metabolic and/or
signal pathways to which the gene or protein belong, and the like),
algorithmic analysis of the compounds to determine critical
features and likely cross-reactivity, and the like. The product
information can take the form of data or information physically
present in the database, hyperlinks to external information sources
(such as a vendor's catalog, for example, supplied via the Internet
or CD-ROM), and the like.
[0631] The database thus preferably contains five main types of
data: gene information, compound information, bioassay information,
product information, and profile information. Gene information
comprises information specific to each included gene, and can
include, for example, the identity and sequence of the gene, one or
more unique identifiers linked to public and/or commercial
databases, its location on a standard array plate, a list of genes
having similar sequences, any known disease associations, any known
compounds that modulate the encoded protein activity, conditions
that modulate expression of the gene or modulate the protein
activity, and the like. Product information comprises information
specific to the available products, and varies depending on the
exact nature of the product, and can include information such as
price, manufacturer, contents, warranty information, availability,
delivery time, distributor, and the like. Bioassay information
comprises information specific to particular compounds (where
available), and can include, for example, results from
high-throughput screening assays, cellular assays, animal and/or
human studies, biochemical assays (including binding assays and
enzymatic assays) and the like. Compound information comprises
information specific to each included compound, such as, for
example, the chemical name(s) and structure of the compound, its
molecular weight, solubility and other physical properties,
proteins that it is known to interact with, the profiles in which
it appears, the genes that are affected by its presence, and
available assays for its activity. Profile information includes,
for example, the conditions under which it was generated
(including, for example, the cell type(s) used, the species used,
temperature and culture conditions, compounds present, time
elapsed, and the like), the genes modulated with reference to a
standard, a list of similar profiles, and the like. The information
is obtained by assimilation of and/or reference to
currently-available databases, and by collecting experimental data.
It should be noted that the gene database, although large, contains
a finite number of records, limited by the number of genes in the
organisms under study. The compound database is potentially
unlimited, as new compounds are made and tested constantly. The
profile database, however, is still larger, as it represents
information regarding the interaction of a very large number of
genes with a potentially infinite number of different compounds,
under a variety of conditions.
[0632] Experimental data is preferably collected using a
high-throughput assay format, capable of examining, for example,
the effects of a plurality of compounds (preferably a large number
of standard compounds, for example 10,000) when administered
individually or as a mixture to a plurality of different cell
types. Assay data collected using a uniform format are more readily
comparable, and provide a more accurate indication of the
differences between, for example, the activity of similar
compounds, or the differences in sensitivity of similar genes.
[0633] The system provides several different ways to access the
information contained within the database. An operator can enter a
test gene expression profile into the system, cause the system to
compare the test profile with stored standard gene expression
profiles in the database, and obtain an output comprising one or
more standard expression profiles that are similar to the test
profile. The standard expression profiles are preferably
accompanied by annotations, for example providing information to
the operator as to the similarity of the test profile to standard
profiles obtained from disease states and/or standard compounds.
The test gene expression profile preferably includes an indication
of the conditions under which the profile is obtained, for example
a representation of a test compound used, and/or the culture
conditions.
[0634] The output preferably further comprises a list of the
organ-specific genes, proteins, or transcripts that are modulated
(up-regulated or down-regulated) in the test profile, as compared
with a pre-established expression value, a pre-selected standard
expression profile, a second test expression profile, or another
pre-set threshold value.
[0635] The output is preferably hyperlinked, so that the operator
can easily switch from, for example, a listing of the similar
standard expression profiles to a listing of the modulated genes in
a selected standard expression profile, or from a gene listed in
the test profile to a list of the standard expression profiles in
which the gene is similarly modulated, or to a list of the standard
compounds (and/or conditions) which appear to modulate the selected
gene. The output can comprise correlation information that
highlights features in common between different genes, targets,
profiles, compounds, assays, and the like, to assist the user in
drawing useful correlations. For example, the output can contain a
list of genes that were modulated in the user's experiment with a
selected compound: if a plurality of the genes are indicated as
associated with liver toxicity, the system can prompt the user that
the compound is associated with a toxic drug signature, and prompt
the user to continue with the next compound. Conversely, the output
could indicate previously unnoticed associations between different
pathways, leading the user to explore a hitherto unknown
connection. The output preferably includes hyperlinks to product
information, encouraging the user to purchase or order one or more
products from a selected vendor, where the product(s) relate
specifically to the focus of the database inquiry and the
correlation information that results, and is presented back to the
user to facilitate hypothesis generation. For example, the output
can provide links to products useful for confirming the apparent
activity of a compound, for measuring biological activity directly,
for assaying the compound for possible side effects, and the like,
prompting the user to select products useful in the next stage of
experimentation.
[0636] The system is preferably provided with an algorithm for
assessing similarity of compounds. Suitable methods for comparing
compounds and determining their morphological similarity include
"3D-MI", as set forth in application U.S. Ser. No. 09/475,413, now
U.S. Pat. No. 6,470,305, incorporated herein by reference in full,
Tanimoto similarity (Daylight Software), and the like. Preferably,
the system can be queried for any compounds that are similar to the
test compound in structure and/or morphology. The output from this
query preferably includes the corresponding standard expression
profiles (or hyperlinks to the corresponding standard expression
profiles), and preferably further includes a listing, description,
or hyperlink to an assay capable of determining the biological
activity of the standard and/or test compound.
[0637] Thus, for example, if the user inputs an experimental
expression profile resulting from incubation of test cells with a
particular experimental compound, the user can obtain an output
comprising an estimate of the quality of the data, an
identification of the genes affected by the compound, a listing of
similar profiles and the conditions under which they were obtained
(for example, the compounds used), and a list of compounds having a
structural similarity. The output can be provided in a hyperlinked
format that permits the user to then investigate and explore the
data. For example, the user can examine which genes are modulated,
and determine whether or not the genes have yet been characterized
as to function or activity, and under what conditions each gene is
modulated in a similar fashion. Alternatively, the user can compare
the profile obtained with the profile of a desired outcome, for
example comparing the profile obtained by incubation of diseased or
infected tissue with a test compound against a profile obtained
from healthy (unperturbed) tissue. Alternatively, the user can
compare the profile with the profiles obtained using standard
compounds, for example using a drug of known activity, mechanism of
action, and specificity, thus determining whether the test compound
operates by a different mechanism, or if by the same mechanism
whether it is more or less active than the standard. Additionally,
the user can compare the structure of the test compound with the
structures of other compounds with similar profiles (to determine
which structural features of the compounds are common, and thus
likely to be important for activity), or can compare the compound's
profile with the profiles obtained from structurally similar
compounds in general.
[0638] The system can be configured as a single, integrated whole,
or can be distributed over a variety of locations. For example, the
system can be provided as a central database/server with
remotely-located access units. The remote access units can be
provided with sufficient system capability to accept and interpret
test gene expression profiles, and to compare the test profiles
with standard gene expression profiles. Remote units can further be
provided with a copy of some or all of the database information.
Optionally, the remote system can be used to upload test gene
expression profiles to the central system to update the central
database, or a "private" database supplementary to the main
database can be stored in or near the remote unit.
[0639] The present invention also generally provides, as noted
above, for the monitoring of the perturbation of the levels of
organ-specific polypeptides and transcripts and is certain
embodiments, secreted organ-specific polypeptides and their
transcripts. Such monitoring can be performed by analysis of tissue
samples, cell samples and biological fluid samples and the like. In
specific embodiments, blood is utilized as for analysis. The
monitoring perturbations in organ-specific protein and transcript
levels can assist in monitoring, diagnosing, imaging, and treating
neurological diseases, immune system related diseases,
cardiovascular diseases, infectious diseases, cancers, and
essentially any disease, state, or indication as the health of each
organ or a collection of organs can be simultaneously
monitored.
[0640] The present invention contemplates the use of blood to
determine the quantitative expression of various organ-specific,
tissue-specific, or cell-specific mRNAs that reflect the
health/disease state of the subject through the use of RT-PCR
analysis (or similar amplification techniques). This entire process
takes about three hours or less. The single drop of blood may also
be used for multiple RT-PCR analyses. It is believed that the
present finding can potentially revolutionize the way that diseases
are detected, diagnosed and monitored because it provides a
non-invasive, simple, highly sensitive and quick screening for
tissue-specific transcripts. The transcripts detected in whole
blood have potential as prognostic or diagnostic markers of
disease, as they reflect disturbances in homeostasis in the human
body. Delineation of the sequences and/or quantitation of the
expression levels of these marker genes by RT-PCR will allow for an
immediate and accurate diagnostic/prognostic test for disease or to
assess the efficacy and monitor a particular therapeutic.
[0641] In one embodiment of the present invention, there is
provided a method for detecting expression of a organ-specific
transcript or protein in blood from a subject, comprising the steps
of: a) quantifying RNA or protein from a subject blood sample; and
b) detecting expression of the protein or gene in the quantified
RNA, wherein the expression of protein or the gene in quantified
RNA indicates the expression of the protein or gene in the subject
blood. An example of the quantifying method is by mass
spectrometry.
[0642] In another embodiment of the present invention, there is
provided a method for detecting expression of one or more genes in
blood from a subject, comprising the steps of: a) obtaining a
subject blood sample; b) extracting RNA from the blood sample; c)
amplifying the RNA; d) generating expressed sequence tags (ESTs)
from the amplified RNA product; and e) detecting expression of the
genes in the ESTs, wherein the expression of the genes in the ESTs
indicates the expression of the genes in the subject blood.
Preferably, the subject is a fetus, an embryo, a child, an adult or
a non-human animal. The genes are non-cancer-associated and
tissue-specific genes. Still preferably, the amplification is
performed by RT-PCR using random sequence primers or gene-specific
primers.
[0643] In still another embodiment of the present invention, there
is provided a method for detecting expression of one or more genes
in blood from a subject, comprising the steps of: a) obtaining a
subject blood sample; b) extracting DNA fragments from the blood
sample; c) amplifying the DNA fragments; and d) detecting
expression of the genes in the amplified DNA product, wherein the
expression of the genes in the amplified DNA product indicates the
expression of the genes in the subject blood.
[0644] In yet another embodiment of the present invention, there is
provided a method for monitoring a course of a therapeutic
treatment in an individual, comprising the steps of: a) obtaining a
blood sample from the individual; b) extracting RNA from the blood
sample; c) amplifying the RNA; d) generating expressed sequence
tags (ESTs) from the amplified RNA product; e) detecting expression
of genes in the ESTs, wherein the expression of the genes is
associated with the effect of the therapeutic treatment; and f)
repeating steps a)-e), wherein the course of the therapeutic
treatment is monitored by detecting the change of expression of the
genes in the ESTs. Such a method may also be used for monitoring
the onset of overt symptoms of a disease, wherein the expression of
the genes is associated with the onset of the symptoms. Preferably,
the amplification is performed by RT-PCR, and the change of the
expression of the genes in the ESTs is monitored by sequencing the
ESTs and comparing the resulting sequences at various time points;
or by performing single nucleotide polymorphism analysis and
detecting the variation of a single nucleotide in the ESTs at
various time points.
[0645] In still yet another embodiment of the present invention,
there is provided a method for diagnosing a disease in a test
subject, comprising the steps of: a) generating a cDNA library for
the disease from a whole blood sample from a normal subject; b)
generating expressed sequence tag (EST) profile from the normal
subject cDNA library; c) generating a cDNA library for the disease
from a whole blood sample from a test subject; d) generating EST
profile from the test subject cDNA library; and e) comparing the
test subject EST profile to the normal subject EST profile, wherein
if the test subject EST profile differs from the normal subject EST
profile, the test subject might be diagnosed with the disease.
[0646] In still yet another embodiment of the present invention,
there is provided a kit for diagnosing, prognosing or predicting a
disease, comprising: a) gene-specific primers; wherein the primers
are designed in such a way that their sequences contain the
opposing ends of two adjacent exons for the specific gene with the
intron sequence excluded; and b) a carrier, wherein the carrier
immobilizes the primer(s). Preferably, the gene-specific primers
are selected from the group consisting of insulin-specific primers,
atrial natriuretic factor-specific primers, zinc finger protein
gene-specific primers, beta-myosin heavy chain gene-specific
primers, amyloid precursor protein gene-specific primers, and
adenomatous polyposis-coli protein gene-specific primers. Such a
kit may be applied to a test subject whole blood sample to
diagnose, prognose or predict a disease by detecting the
quantitative expression levels of specific genes associated with
the disease in the test subject and then comparing to the levels of
same genes expressed in a normal subject. Such a kit may also be
used for monitoring a course of therapeutic treatment or monitoring
the onset of overt symptoms of a disease.
[0647] In yet another embodiment of the present invention, there is
provided a kit for diagnosing, prognosing or predicting a disease,
comprising: a) probes derived from a whole blood sample for a
specific disease; and b) a carrier, wherein the carrier immobilizes
the probes. Such a kit may be applied to a test subject whole blood
sample to diagnose, prognose or predict a disease by detecting the
quantitative expression levels of specific genes associated with
the disease in the test subject and then comparing to the levels of
same genes expressed in a normal subject. Such a kit may also be
used for monitoring a course of therapeutic treatment or monitoring
the onset of overt symptoms of a disease.
[0648] Furthermore, the present invention provides a cDNA library
specific for a disease, wherein the cDNA library is generated from
whole blood samples.
[0649] In one embodiment of the present invention, there is a
method of identifying one or more genetic markers for a disease,
wherein each of said one or more genetic markers corresponds to a
gene transcript, comprising the steps of: a) determining the level
of one or more gene transcripts expressed in blood obtained from
one or more individuals having a disease, wherein each of said one
or more transcripts is expressed by a gene that is a candidate
marker for disease; and b) comparing the level of each of said one
or more gene transcripts from said step a) with the level of each
of said one or more genes transcripts in blood obtained from one or
more individuals not having a disease, wherein those compared
transcripts which display differing levels in the comparison of
step b) are identified as being genetic markers for a disease.
[0650] In another embodiment of the present invention, there is a
method of identifying one or more genetic markers for a disease,
wherein each of said one or more genetic markers corresponds to a
gene transcript, comprising the steps of: a) determining the level
of one or more gene transcripts expressed in blood obtained from
one or more individuals having a disease, wherein each of said one
or more transcripts is expressed by a gene that is a candidate
marker for a disease; and b) comparing the level of each of said
one or more gene transcripts from said step a) with the level of
each of said one or more genes transcripts in blood obtained from
one or more individuals having a disease, wherein those compared
transcripts which display the same levels in the comparison of step
b) are identified as being genetic markers for a disease.
[0651] In another embodiment of the present invention, there is a
method of identifying one or more genetic markers of a stage of a
disease progression or regression, wherein each of said one or more
genetic markers corresponds to a gene transcript, comprising the
steps of: a) determining the level of one or more gene transcripts
expressed in blood obtained from one or more individuals having a
stage of a disease, wherein said one or more individuals are at the
same progressive or regressive stage of a disease, and wherein each
of said one or more transcripts is expressed by a gene that is a
candidate marker for determining the stage of progression or
regression of a disease, and; b) comparing the level of each of
said one or more gene transcripts from said step a) with the level
of each of said one or more genes transcripts in blood obtained
from one or more individuals who are at a progressive or regressive
stage of a disease distinct from that of said one or more
individuals of step a), wherein those compared transcripts which
display differing levels in the comparison of step b) are
identified as being genetic markers for the stage of progression or
regression of a disease.
[0652] In another embodiment of the present invention, there is a
method of identifying one or more genetic markers of a stage of a
disease progression or regression, wherein each of said one or more
genetic markers corresponds to a gene transcript, comprising the
steps of: a) determining the level of one or more gene transcripts
expressed in blood obtained from one or more individuals having a
stage of a disease, wherein said one or more individuals are at the
same progressive or regressive stage of a disease, and wherein each
of said one or more transcripts is expressed by a gene that is a
candidate marker for determining the stage of progression or
regression of a disease, and b) comparing the level of each of said
one or more gene transcripts from said step a) with the level of
each of said one or more genes transcripts in blood obtained from
one or more individuals who are at a progressive or regressive
stage of a disease identical to that of said one or more
individuals of step a), wherein those compared transcripts which
display the same levels in the comparison of step b) are identified
as being genetic markers for the stage of progression or regression
of a disease.
[0653] In another embodiment of the present invention, there is a
method of diagnosing or prognosing a disease in an individual,
comprising the steps of: a) determining the level of one or more
gene transcripts in blood obtained from said individual suspected
of having a disease, and b) comparing the level of each of said one
or more gene transcripts in said blood according to step a) with
the level of each of said one or more gene transcripts in blood
from one or more individuals not having a disease, wherein
detecting a difference in the levels of each of said one or more
gene transcripts in the comparison of step b) is indicative of a
disease in the individual of step a).
[0654] In another embodiment of the present invention, there is a
method of diagnosing or prognosing a disease in an individual,
comprising the steps of: a) determining the level of one or more
organ-specific gene transcripts or organ-specific proteins in blood
obtained from said individual suspected of having a disease, and b)
comparing the level of each of said one or more transcripts or
protein in said blood according to step a) with the level of each
of said one or more transcripts or protein in blood from one or
more individuals having a disease, wherein detecting the same
levels of each of said one or more transcripts or proteins in the
comparison of step b) is indicative of a disease in the individual
of step a).
[0655] In another embodiment of the present invention, there is a
method of determining a stage of disease progression or regression
in an individual having a disease, comprising the steps of: a)
determining the level of one or more organ-specific gene
transcripts or organ-specific proteins in blood obtained from said
individual having a disease, and b) comparing the level of each if
said one or more organ-specific gene transcripts or organ-specific
proteins in said blood according to step a) with the level of each
of said one or more organ-specific gene transcripts or
organ-specific proteins in blood obtained from one or more
individuals who each have been diagnosed as being at the same
progressive or regressive stage of a disease, wherein the
comparison from step b) allows the determination of the stage of a
disease progression or regression in an individual.
[0656] Further embodiments of the methods described herein include
embodiments comprising a further step of isolating RNAfrom said
blood samples, and embodiments comprising determining the level of
each of said one or more gene transcripts comprising quantitative
RT-PCR (QRT-PCR), wherein said one or more transcripts are from
step a) and/or step b) of said methods. Further embodiments of
these methods include embodiments wherein said QRT-PCR comprises
primers which hybridize to one or more transcripts or the
complement thereof, wherein said one or more transcripts are from
step a) and/or step b) of said methods, embodiments wherein said
primers are 15-25 nucleotides in length, and embodiments wherein
said primers hybridize to one or more of the sequences of any one
of Tables 1-32, 36-45 and 47-79, or the complement thereof. Further
embodiments of the methods described in the previous eight
paragraphs include embodiments wherein the step of determining the
level of each of said one or more gene transcripts comprises
hybridizing a first plurality of isolated nucleic acid molecules
that correspond to said one or more transcripts to an array
comprising a second plurality of isolated nucleic acid molecules,
wherein in one embodiment said first plurality of isolated nucleic
acid molecules comprises RNA, DNA, cDNA, PCR products or ESTs,
wherein in one embodiment said array comprises a plurality of
isolated nucleic acid molecules comprising RNA, DNA, cDNA, PCR
products or ESTs, wherein in one embodiment said array comprises
two or more of the genetic markers of said methods, wherein in one
embodiment said array comprises a plurality of nucleic acid
molecules that correspond to genes of the human genome.
[0657] In another embodiment of the present invention, kits or
panels comprise a plurality of nucleic acid molecules or protein
sequences that correspond to two or more sequences from each of any
one of Tables 1-32, 36-45 and 47-79.
[0658] In another embodiment of the present invention, there is an
array which comprises a plurality of nucleic acid molecules or
protein-binding agents (such as immunoglobulins and fragments
thereof) that correspond or specifically bind to two or more
sequences from each of any one of Tables 1-32, 36-45 and 47-79.
[0659] In another embodiment of the present invention, there is a
kit for monitoring a course of therapeutic treatment of a disease,
comprising a) two gene-specific priming means designed to produce
double stranded DNA complementary to a gene selected group
consisting of any one of Tables 1-32, 36-45 and 47-79; wherein said
first priming means contains a sequence which can hybridize to RNA,
cDNA or an EST complementary to said gene to create an extension
product and said second priming means capable of hybridizing to
said extension product; b) an enzyme with reverse transcriptase
activity c) an enzyme with thermostable DNA polymerase activity and
d) a labeling means; wherein said primers are used to detect the
quantitative expression levels of said gene in a test subject.
[0660] In another embodiment of the present invention, there is a
kit for monitoring progression or regression of a disease,
comprising: a) two gene-specific priming means designed to produce
double stranded DNA complementary to a gene selected group
consisting of any one of Tables 1-32, 36-45 and 47-79; wherein said
first priming means contains a sequence which can hybridize to RNA,
cDNA or an EST complementary to said gene to create an extension
product and said second priming means capable of hybridizing to
said extension product; b) an enzyme with reverse transcriptase
activity c) an enzyme with thermostable DNA polymerase activity and
d) a labeling means; wherein said primers are used to detect the
quantitative expression levels of said gene in a test subject.
[0661] In another embodiment of the present invention, there is a
plurality of nucleic acid molecules or polypeptide molecules that
identify or correspond to two or more sequences from any one of
Tables 1-32, 36-45 and 47-79.
[0662] It would be readily understood by review of the instant
specification that while some methods are described as gene or
nucleic acid based or polypeptide based, that all such methods
would be readily interchangeable. Accordingly, where a method is
described that could use a polypeptide for detection of another
polypeptide in place of nucleic acid to nucleic acid detection and
vice versa, such interchangeability is explicitly considered to be
a part of the invention described herein. Likewise, wherein blood
is described as the prototypic biological component for analysis,
it should be understood that any cell sample, tissue sample, or
biological fluid sample may be used interchangeably therewith.
[0663] As used herein, a disease of the invention includes, but is
not limited to, blood disorders, blood lipid disease, autoimmune
disease, arthritis (including osteoarthritis, rheumatoid arthritis,
lupus, allergies, juvenile rheumatoid arthritis and the like), bone
or joint disorder, a cardiovascular disorder (including heart
failure, congenital heart disease; rheumatic fever, valvular heart
disease; corpulmonale, cardiomyopathy, myocarditis, pericardial
disease; vascular diseases such as atherosclerosis, acute
myocardial infarction, ischemic heart disease and the like),
obesity, respiratory disease (including asthma, pneumonitis,
pneumonia, pulmonary infections, lung disease, bronchiectasis,
tuberculosis, cystic fibrosis, interstitial lung disease, chronic
bronchitis emphysema, pulmonary hypertension, pulmonary
thromboembolism, acute respiratory distress syndrome and the like),
hyperlipidemias, endocrine disorder, immune disorder, infectious
disease, muscle wasting and whole body wasting disorder,
neurological disorders (including migraines, seizures, epilepsy,
cerebrovascular diseases, alzheimers, dementia, Parkinson's, ataxic
disorders, motor neuron diseases, cranial nerve disorders, spinal
cord disorders, meningitis and the like) including
neurodegenerative and/or neuropsychiatric diseases and mood
disorders (including schizophrenia, anxiety, bipolar disorder;
manic depression and the like, skin disorder, kidney disease,
scleroderma, stroke, hereditary hemorrhage telangiectasia,
diabetes, disorders associated with diabetes (e.g., PVD),
hypertension, Gaucher's disease, cystic fibrosis, sickle cell
anemia, liver disease, pancreatic disease, eye, ear, nose and/or
throat disease, diseases affecting the reproductive organs,
gastrointestinal diseases (including diseases of the colon,
diseases of the spleen, appendix, gall bladder, and others) and the
like. For further discussion of human diseases, see Mendelian
Inheritance in Man: A Catalog of Human Genes and Genetic Disorders
by Victor A. McKusick (12th Edition (3 volume set) June 1998, Johns
Hopkins University Press, ISBN: 0801857422) and Harrison's
Principles of Internal Medicine by Braunwald, Fauci, Kasper,
Hauser, Longo, & Jameson (15th Edition, 2001), the entirety of
which is incorporated herein.
[0664] In another embodiment of the invention, a disease refers to
an immune disorder, such as those associated with overexpression of
a gene or expression of a mutant gene (e.g., autoimmune diseases,
such as diabetes mellitus, arthritis (including rheumatoid
arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic
arthritis), multiple sclerosis, encephalomyelitis, myasthenia
gravis, systemic lupus erythematosis, autoimmune thyroiditis,
dermatitis (including atopic dermatitis and eczematous dermatitis),
psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer,
iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis,
asthma, allergic asthma, cutaneous lupus erythematosus,
scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal
reactions, erythema nodosum leprosum, autoimmune uveitis, allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy,
idiopathic bilateral progressive sensorineural hearing, loss,
aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia,
polychondritis, Wegener's granulomatosis, chronic active hepatitis,
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'
disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior,
and interstitial lung fibrosis), graft-versus-host disease, cases
of transplantation, and allergy.
[0665] In another embodiment, a disease of the invention is a
cellular proliferative and/or differentiative disorder that
includes, but is not limited to, cancer e.g., carcinoma, sarcoma or
other metastatic disorders and the like. As used herein, the term
"cancer" refers to cells having the capacity for autonomous growth,
i.e., an abnormal state of condition characterized by rapidly
proliferating cell growth. "Cancer" is meant to include all types
of cancerous growths or oncogenic processes, metastatic tissues or
malignantly transformed cells, tissues, or organs, irrespective of
histopathologic type or stage of invasiveness. Examples of cancers
include but are nor limited to solid tumors and leukemias,
including: apudoma, choristoma, branchioma, malignant carcinoid
syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal
cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumour, in situ,
Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell,
papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and
transitional cell), histiocytic disorders, leukaemia (e.g., B cell,
mixed cell, null cell, T cell, T-cell chronic, HTLV-II-associated,
lymphocytic acute, lymphocytic chronic, mast cell, and myeloid),
histiocytosis malignant, Hodgkin disease, immunoproliferative
small, non-Hodgkin lymphoma, plasmacytoma, reticuloendotheliosis,
melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma,
fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma,
mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing
sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma,
chordoma, craniopharyngioma, dysgerminoma, hamartoma, mesenchymoma,
mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma,
teratoma, thymoma, trophoblastic tumour, adeno-carcinoma, adenoma,
cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma,
cystadenoma, granulosa cell tumour, gynandroblastoma, hepatoma,
hidradenoma, islet cell tumour, Leydig cell tumour, papilloma,
Sertoli cell tumour, theca cell tumour, leiomyoma, leiomyosarcoma,
myoblastoma, mymoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma,
ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma,
neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma,
neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma,
angiolymphoid hyperplasia with eosinophilia, angioma sclerosing,
angiomatosis, glomangioma, hemangioendothelioma, hemangioma,
hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma,
lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma,
cystosarcoma, phyllodes, fibrosarcoma, hemangiosarcoma,
leimyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma,
myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma,
sarcoma (e.g., Ewing, experimental, Kaposi, and mast cell),
neoplasms (e.g., bone, breast, digestive system, colorectal, liver,
pancreatic, pituitary, testicular, orbital, head and neck, central
nervous system, acoustic, pelvic respiratory tract, and
urogenital), neurofibromatosis, and cervical dysplasia, and other
conditions in which cells have become immortalized or
transformed.
[0666] In another embodiment, a disease of the invention includes
but is not limited to a condition wherein said condition is
reflective of the state of a particular individual, whether said
state is a physical, emotional or psychological state, said state
resulting from the progression of time, treatment, environmental
factors or genetic factors.
[0667] When comparing two or more samples for differences, results
are reported as statistically significant when there is only a
small probability that similar results would have been observed if
the tested hypothesis (i.e., the genes are not expressed at
different levels) were true. A small probability can be defined as
the accepted threshold level at which the results being compared
are considered significantly different. The accepted lower
threshold is set at, but not limited to, 0.05 (i.e., there is a 5%
likelihood that the results would be observed between two or more
identical populations) such that any values determined by
statistical means at or below this threshold are considered
significant.
[0668] When comparing two or more samples for similarities, results
are reported as statistically significant when there is only a
small probability that similar results would have been observed if
the tested hypothesis (i.e., the genes are not expressed at
different levels) were true. A small probability can be defined as
the accepted threshold level at which the results being compared
are considered significantly different. The accepted lower
threshold is set at, but not limited to, 0.05 (i.e., there is a 5%
likelihood that the results would be observed between two or more
identical populations) such that any values determined by
statistical means above this threshold are not considered
significantly different and thus similar.
[0669] Identification of genes differentially expressed in blood
samples from patients with disease as compared to healthy patients
or as compared to patients without said disease is determined by
statistical analysis of the gene expression profiles from healthy
patients or patients without disease compared to patients with
disease using the Wilcox Mann Whitney rank sum test. Other
statistical tests can also be used, see for example (Sokal and
Rohlf (1987) Introduction to Biostatistics 2nd edition, WH Freeman,
New York), which is incorporated herein in their entirety.
[0670] In order to facilitate ready access, e.g., for comparison,
review, recovery and/or modification, the expression profiles of
patients with disease and/or patients without disease or healthy
patients can be recorded in a database, whether in a relational
database accessible by a computational device or other format, or a
manually accessible indexed file of profiles as photographs,
analogue or digital imaging, readouts spreadsheets etc. Typically
the database is compiled and maintained at a central facility, with
access being available locally and/or remotely.
[0671] As would be understood by a person skilled in the art,
comparison as between the expression profile of a test patient with
expression profiles of patients with a disease, expression profiles
of patients with a certain stage or degree of progression of said
disease, without said disease, or a healthy patient so as to
diagnose or prognose said test patient can occur via expression
profiles generated concurrently or non concurrently. It would be
understood that expression profiles can be stored in a database to
allow said comparison.
[0672] As additional test samples from test patients are obtained,
through clinical trials, further investigation, or the like,
additional data can be determined in accordance with the methods
disclosed herein and can likewise be added to a database to provide
better reference data for comparison of healthy and/or non-disease
patients and/or certain stage or degree of progression of a disease
as compared with the test patient sample. These and other methods,
including those described in the art (e.g., U.S. Patent Application
Pub No. 20060134637) can be used in the context of the sequences
disclosed.
Business Methods
[0673] A further embodiment of the present invention comprises
business methods for manufacturing one or more of the detection
reagents, panels, arrays as described herein as well as providing
diagnostic services for analyzing and/or comparing organ-specific
fingerprints or individual proteins (or nucleic acid molecules)
from a subject with one, two or more organ-specific proteins or
nucleic acid molecules described herein, identifying disease
fingerprints or organ-specific proteins or nucleic acid molecules
that vary or become present with disease, identifying fingerprints
or proteins or nucleic acid molecule levels perturbed from normal,
providing manufacturers of genomics devices the use of the
detection reagents, panels, arrays, organ-specific fingerprints or
specific organ-specific proteins or nucleic acid probes for nucleic
acid molecules encoding the same described herein to develop
diagnostic devices, where the genomics device includes any device
that may be used to define differences in a sample between the
normal and disturbed state resulting from one or more effects,
providing manufacturers of proteomics devices the use of the
detection reagents, panels, arrays, organ-specific proteins
described herein to develop diagnostic devices, where the
proteomics device includes any device that may be used to define
differences in a sample between the normal and disturbed state
resulting a disease, disorder or therapy, providing manufacturers
of imaging devices detection reagents, panels, arrays, lateral flow
devices, organ-specific proteins or nucleic acid molecules or
probes thereto described herein to develop diagnostic devices,
where the proteomics devices include any device that may be used to
define differences in a blood sample between the normal and
disturbed state resulting from disease, drug side-effects, or
therapeutic interventions, providing manufacturers of molecular
imaging devices the use of the detection reagents, panels, arrays,
blood fingerprints or transcriptomes described herein to develop
diagnostic devices, where the proteomics device includes any device
that may be used to define differences in a blood sample between
the normal and disturbed state and marketing to healthcare
providers the benefits of using the detection reagents, panels,
arrays, and diagnostic services of the present invention to enhance
diagnostic capabilities and thus, to better treat patients.
[0674] Also provided is an aspect of the invention to utilize
databases to store data and analysis of panels and
organ/tissue-specific sets and individual components thereof for
certain ethnic populations, genders, etc. and for analysis over a
lifetime for individuals based upon the data from millions or more
individuals. In addition, the present invention contemplates the
storage an access to such information via an appropriate secured
and private setting wherein HIPAA standards are followed.
[0675] Another aspect of the invention relates to a method for
conducting a business, which includes: (a) manufacturing one or
more of the detection reagents, panels, arrays, (b) providing
services for analyzing organ-specific molecular blood fingerprints
and (c) marketing to healthcare providers the benefits of using the
detection reagents, panels, arrays, and services of the present
invention to enhance capabilities to detect disease or disease
progression and thus, to better treat patients.
[0676] Another aspect of the invention relates to a method for
conducting a business, comprising: (a) providing a distribution
network for selling the detection reagents, panels, arrays,
diagnostic services, and access to organ-specific molecular blood
fingerprint databases (b) providing instruction material to
physicians or other skilled artisans for using the detection
reagents, panels, arrays, and organ-specific molecular blood
fingerprint databases to improve the ability to detect disease,
analyze disease progression, or stratify patients.
[0677] For instance, the subject business methods can include an
additional step of providing a sales group for marketing the
database, or panels, or arrays, to healthcare providers.
[0678] Another aspect of the invention relates to a method for
conducting a business, comprising: (a) preparing one or more normal
organ-specific molecular blood fingerprints and (b) licensing, to a
third party, the rights for further development and sale of panels,
arrays, and information databases related to the organ-specific
molecular blood fingerprints of (a).
[0679] The business methods of the present application relate to
the commercial and other uses, of the methodologies, panels,
arrays, organ-specific proteins (e.g., including secreted
organ-specific proteins and panels thereof), organ-specific
molecular blood fingerprints, and databases comprising identified
fingerprints of the present invention. In one aspect, the business
method includes the marketing, sale, or licensing of the present
invention in the context of providing consumers, i.e., patients,
medical practitioners, medical service providers, and
pharmaceutical distributors and manufacturers, with all aspects of
the invention described herein, (e.g., the methods for identifying
organ-specific secreted proteins, detection reagents for such
proteins, molecular blood fingerprints, etc., as provided by the
present invention).
[0680] In a particular embodiment of the present invention, a
business method or diagnostic method relating to providing
information related to organ-specific proteins (nucleic acids
encoding the same), a plurality thereof, or a fingerprint of a
plurality (e.g., levels of the plurality of organ-specific secreted
proteins that make up a given fingerprint), method for determining
organ-specific protein (or transcripts encoding the same) or levels
thereof or fingerprints of the same and sale of such panels. In a
specific embodiment, that method may be implemented through the
computer systems of the present invention. For example, a user
(e.g. a health practitioner such as a physician or a diagnostic
laboratory technician) may access the computer systems of the
present invention via a computer terminal and through the Internet
or other means. The connection between the user and the computer
system is preferably secure.
[0681] In practice, the user may input, for example, information
relating to a patient such as the patient's disease state and/or
drugs that the patient is taking, e.g., levels determined for the
organ-specific proteins of interest or that make up a given
molecular blood fingerprint using a panel or array of the present
invention. The computer system may then, through the use of the
resident computer programs, provide a diagnosis, detect changes in
disease states, stratify patients, or determination of drug
side-effects that fits with the input information by matching the
parameters of particular protein or panel thereof (e.g., levels of
the proteins present in the blood as detected using a particular
panel or array of the present invention) with a database of
fingerprints.
[0682] A computer system in accordance with a preferred embodiment
of the present invention may be, for example, an enhanced IBM
AS/400 mid-range computer system. However, those skilled in the art
will appreciate that the methods and apparatus of the present
invention apply equally to any computer system, regardless of
whether the computer system is a complicated multi-user computing
apparatus or a single user device such as a personal computer or
workstation. Computer systems suitably comprise a processor, main
memory, a memory controller, an auxiliary storage interface, and a
terminal interface, all of which are interconnected via a system
bus. Note that various modifications, additions, or deletions may
be made to the computer system within the scope of the present
invention such as the addition of cache memory or other peripheral
devices.
[0683] The processor performs computation and control functions of
the computer system, and comprises a suitable central processing
unit (CPU). The processor may comprise a single integrated circuit,
such as a microprocessor, or may comprise any suitable number of
integrated circuit devices and/or circuit boards working in
cooperation to accomplish the functions of a processor.
[0684] In a preferred embodiment, the auxiliary storage interface
allows the computer system to store and retrieve information from
auxiliary storage devices, such as magnetic disk (e.g., hard disks
or floppy diskettes) or optical storage devices (e.g., CD-ROM). One
suitable storage device is a direct access storage device (DASD). A
DASD may be a floppy disk drive that may read programs and data
from a floppy disk. It is important to note that while the present
invention has been (and will continue to be) described in the
context of a fully functional computer system, those skilled in the
art will appreciate that the mechanisms of the present invention
are capable of being distributed as a program product in a variety
of forms, and that the present invention applies equally regardless
of the particular type of signal bearing media to actually carry
out the distribution. Examples of signal bearing media include:
recordable type media such as floppy disks and CD ROMS, and
transmission type media such as digital and analog communication
links, including wireless communication links.
[0685] The computer systems of the present invention may also
comprise a memory controller, through use of a separate processor,
which is responsible for moving requested information from the main
memory and/or through the auxiliary storage interface to the main
processor. While for the purposes of explanation, the memory
controller is described as a separate entity, those skilled in the
art understand that, in practice, portions of the function provided
by the memory controller may actually reside in the circuitry
associated with the main processor, main memory, and/or the
auxiliary storage interface.
[0686] Furthermore, the computer systems of the present invention
may comprise a terminal interface that allows system administrators
and computer programmers to communicate with the computer system,
normally through programmable workstations. It should be understood
that the present invention applies equally to computer systems
having multiple processors and multiple system buses. Similarly,
although the system bus of the preferred embodiment is a typical
hardwired, multidrop bus, any connection means that supports
bidirectional communication in a computer-related environment could
be used.
[0687] The main memory of the computer systems of the present
invention suitably contains one or more computer programs relating
to the organ-specific molecular blood fingerprints and an operating
system. Computer program is used in its broadest sense, and
includes any and all forms of computer programs, including source
code, intermediate code, machine code, and any other representation
of a computer program. The term "memory" as used herein refers to
any storage location in the virtual memory space of the system. It
should be understood that portions of the computer program and
operating system may be loaded into an instruction cache for the
main processor to execute, while other files may well be stored on
magnetic or optical disk storage devices. In addition, it is to be
understood that the main memory may comprise disparate memory
locations.
[0688] As should be clear to the skilled artisan from the above,
the present invention provides databases, readable media with
executable code, and computer systems containing information
comprising predetermined normal serum levels of organ-specific
proteins that make up organ-specific protein sets. Further, the
present invention provides databases of information comprising
disease-associated organ-specific proteins, nucleic acid molecules
encoding the same, as well as panels and in some embodiments,
levels thereof.
[0689] Throughout this disclosure, various aspects of this
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0690] All of the U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety. Moreover, all
numerical ranges utilized herein explicitly include all integer
values within the range and selection of specific numerical values
within the range is contemplated depending on the particular use.
Further, the following examples are offered by way of illustration,
and not by way of limitation.
EXAMPLES
Example 1
Identification of Organ-Specific Proteins by Analysis of MPSS
Data
[0691] This example shows the identification of organ-specific
protein sets from 32 normal, healthy organs.
1. Normalized MPSS Dataset
[0692] The normalized MPSS data used in this study was previously
described (Jongeneel, et al., Genome (2005), 15:1007-1014) but
re-annotated by Solexa (Hayward, Calif., USA) to the new UniGene
database (web address: ftp colon double slash ftp dot ncbi dot nih
dot gov/repository/UniGene/Homo sapiens/, build 184). The data
contained a total of 391,669 MPSS sequence tags, their gene
annotation, their expression levels in 32 different tissues as
specified by their transcription per million (tpm) and the
associated standard deviation (SD), and information on the sequence
tags such as significance, selected step, class, and global
replication. This dataset was used to identify organ-specific
proteins as described below.
Identification of Organ-Specific MPSS Tags
[0693] Assume the expression (in tpm) and the associated SD of a
MPSS sequence tag in the 32 tissues were {(X.sub.i,.sigma..sub.i)},
where i=1, 2, . . . , 32 represents individual tissues. Assume the
tag had the highest expression levels in tissue m where the
expression and the SD were (X.sub.m,.sigma..sub.m). We then applied
three rules to determine whether the tag was specific to tissue m
as follows:
[0694] i) The expression of the tag in tissue m was above a
minimal, estimated noise levels, i.e.,
X.sub.m.gtoreq.5. (1)
[0695] ii) The expression of the tag in tissue m was well above the
expression of the tag in all other tissues. More specifically, we
first calculated the mean expression of the tag in the other 31
tissues (except tissue m) as
X _ = 1 N i .noteq. m X i , ( 2 ) ##EQU00008##
[0696] the associated standard error as
.sigma. X _ = 1 N i .noteq. m .sigma. i 2 , ( 3 ) ##EQU00009##
[0697] and the corresponding SD as
s = 1 N - 1 i .noteq. m ( X i - X _ ) 2 + 1 N i .noteq. m .sigma. i
2 , ( 4 ) ##EQU00010##
[0698] where N=31.
[0699] We then evaluated the significance that the expression of
the tag in tissue m was above the expression of the tag in other
tissues as
p dis = 1 2 erfc ( X m - X _ 2 ( s 2 + .sigma. m 2 + .sigma. X _ 2
) ) . ( 5 ) ##EQU00011##
[0700] For the tag to be specific to tissue m, we required that
p.sub.dis.ltoreq.10.sup.-3. (6)
[0701] iii) The specificity f of the tag in tissue m had to be well
above a pre-selected cutoff value f.sub.0. More precisely, we
defined the specificity of the tag in tissue m as
f = X m i X i . ( 7 ) ##EQU00012##
[0702] and evaluated the associated SD as
.sigma. f = f X m ( 1 - f ) 2 .sigma. m 2 + f 2 i .noteq. m .sigma.
i 2 . ( 8 ) ##EQU00013##
[0703] The significance that f was above f.sub.0 is then given
by
p spc = 1 2 erfc ( f - f 0 2 .sigma. f ) . ( 9 ) ##EQU00014##
[0704] Nine different values of f.sub.0 and p.sub.spc were applied
in determining organ-specific MPSS tags, ranging from the most
stringent condition (f.sub.0=1 and p.sub.spc.ltoreq.10.sup.-3) to
the least stringent condition (f.sub.0=0.5 and
p.sub.spc.ltoreq.0.1): See Tables 1-32 and 36-45. However, in most
cases, we required that
p.sub.spc.ltoreq.10.sup.-3. (10)
[0705] The stringency for each organ-specific transcript is noted
in Tables 1-32 and 36-45. (see column marked "Specificity")
[0706] When we applied rules i)-iii) (Eqs. (1), (6) and (10) with
f.sub.0=0.8) to the 391,669 MPSS sequence tags, we identified a
total of 17,638 organ-specific tags. The number of organ-specific
tags varies with the selected values of f.sub.0 and p.sub.spc: See
Tables 1-32 and 36-45.
Mapping Organ-Specific MPSS Tags to Corresponding Proteins
[0707] MPSS tags were annotated to gene accession numbers in the
UniGene database by Solexa. In cases where MPSS tags were mapped to
multiple genes in the UniGene database, Solexa intentionally
annotated the MPSS tags only to genes having the longest sequences.
A script was developed to annotate the MPSS tags to all genes
containing the MPSS tags. A separate script was then developed to
map gene accession numbers in the UniGene database to the
corresponding gene accessions, protein accessions and protein
sequences in the RefSeq database (web address: ftp colon double
slash ftp dot ncbi dot gov slash refseq slash H sapiens/). In this
way, organ-specific tags were mapped to corresponding proteins. In
some cases multiple MPSS tags may be mapped to the same proteins.
In such cases only the MPSS tag having the most abundant expression
was kept for each organ-specific protein. The organ-specific
proteins identified are provided in Tables 1-32 and 36-45.
Information on each protein includes gene name, gene accession
number, protein accession number, summary description of the
protein, predicted protein localization (as described further
below), corresponding MPSS tag, the class of the MPSS tag, the
expression of the tag in the particular tissue (count), the
specificity of the tag in the particular tissue (as described
above), and the number of peptides identified on the protein by
HUPO (as described further below). The amino acid sequences
corresponding to the organ-specific proteins and the
polynucleotides encoding the proteins are provided in the sequence
listing. Sequences corresponding to the MPSS tags as shown in
Tables 1-32 and 36-45 are also provided. Sequences corresponding to
peptides previously identified by mass spectrometry that map to
organ-specific proteins described in Tables 43, 44 and 45 are shown
in those Tables and are provided in the sequence listing.
Prediction of Protein Localization
[0708] Protein sequences in the RefSeq database were used to
predict protein localization using TMHMM (server 2.0, http colon
double slash www dot cbs dot dtu dot dk/services/TMHMM/), which
applies hidden Markov model to predict protein transmembrane
domains SignalP (server 3.0, http colon double slash www dot cbs
dot dtu dot dk/services/SignalP/), which applies both artificial
neural network and hidden Markov model to predict the presence and
the location of signal peptide cleavage sites for classical
(N-terminus lead) secreted proteins. This was performed
computationally using a combination of hidden Markov model (HMM)
algorithms (Krogh et al., J Mol Biol (2001) 305:567) and
transmembrane (TM) region predictions from a commercial version of
the TMHMM algorithm. The subcellular localizations of proteins were
categorized as follows: a) extracellular-proteins, (including
secreted proteins) which contain predicted signal peptides and no
predicted transmembrane segments; c) transmembrane-proteins, which
contain predicted transmembrane segments; or d) other which
includes intracellular-proteins, which contain neither predicted
signal peptides nor predicted transmembrane regions was then used
to combine the outputs of the two programs into protein
localization prediction, which is outlined in Table 33. The
localization prediction for all organ-specific proteins is
summarized in Table 33.
TABLE-US-00002 TABLE 33 Rules used to combine outputs of TMHMM and
SignalP for the prediction of protein localization TM domains
Secretion Cleavage Localization (TMHMM) (SignalP) (SignalP)
prediction >1 Transmembrane 1 N Transmembrane 1 (not Y Y
Transmembrane cleaved) 1 Y Y Secreted (cleaved) 0, 1 Y N Anchored 0
Y Y Secreted 0 N Other
Overlap with Identified Human Plasma Proteins
[0709] The Plasma Proteome Project of the Human Proteome
Organization (HUPO) released a list of human plasma proteins
including the number of identified peptides on each protein (Omenn,
et al. Proteomics. (2005):3226-45). These proteins were specified
by protein IDs in the International Protein Index (IPI) database
(web address: ftp colon double slash ftp dot ebi dot ac dot uk
slash pub/databases/IPI/current/ipi dot HUMAN dot fasta dot gz). A
list for mapping protein IPI IDs to protein RefSeq accession
numbers was also provided. Overlap between the organ-specific
proteins and the HUPO plasma proteins were analyzed. The identified
overlap is shown in Tables 1-32. Further classification by class
and ranks were used to formulate statistical tests to determine
significant genes (Stolovitzky et al, PNAS (2005) 1402-1407).
Example 2
Identification of Organ-Specific Proteins in Human Serum Using Mass
Spectrometry
[0710] This experiment demonstrates the process of identifying
organ-specific proteins in a normal sample of blood serum from a
healthy, human volunteer. For normal control serum, venous blood
samples were drawn from a fasted, human volunteer. Samples were
collected with minimal stasis in evacuated serum separator tubes.
After at least 30 min, but within 2 hours, the tubes were
centrifuged at 23.degree. C. for 15 minutes at 1,200 g and serum
was stored in plastic vials at -80.degree. C. To reduce sample
complexity, plasma was passed over a column containing antibodies
to the most abundant proteins. In this example, an affinity column
was used to remove albumin, IgG, IgA, anti-trypsin, transferrin,
and haptoglobin; however, affinity columns with an expanded
repertoire could also be used. Since most proteins found on the
cell surface or secreted from cells are glycoproteins, and can be
isolated via the glycopeptide capture method, proteins were further
enriched and identified by specifically capturing N-linked
glycopeptides from plasma.
[0711] A recently developed mass spectrometry-based screening
technology provided specific targets--glycoproteins in a plasma
sample for identification and quantification. The glycopeptides
isolated from plasma and tissues were analyzed by MALDI-TOF/TOF
(ABI 4800 Proteomics Analyzer, Applied Biosystems) after front-end
separation of peptides using strong cation exchange fractionation
followed by reversed phase chromatography. The advantage of this
platform is its high mass accuracy, resolution, sensitivity, and
the ability to do targeted MS/MS analysis on peptides of interest.
Since the separation is performed off-line, more time is available
for deeper interrogation of the observed tryptic peptides. Multiple
plates can also be spotted and analyzed by MALDI-TOF/TOF to
increase the depth of proteomic coverage. This platform will also
be used in the direct follow up analysis of potential peptides
during the comparison of cancer and control sera using heavy
isotope labeled synthetic peptide standards.
HPLC and Maldi Plate Spotting
[0712] Up to 20 ug total peptide was loaded onto a 150 mm.times.0.3
mm poly sulfoethyl A column, and peptides were eluted using five to
ten steps of increasing salt concentration. Up to 2 ug total
peptide that was bumped from the strong cation exchange column was
trapped onto a 250 micron.times.3 cm trap column self-packed with 5
micron particle Vydac C8 (#208MS54). The Eksigent HPLC and
autosampler (Model: NanoLC-2D, Dublin, Calif.) uses a 10 ul sample
loop. After capturing the strong cation exchange eluate, the trap
column was brought in-line with a 100 micron ID.times.20 cm
resolving column that was self-packed with 5 micron particle Vydac
C18 (#218MS54) at a flow rate of 500 nanoliters per minute using
0.1% TFA and 2% acetonitrile as solvent A, and 0.08% TFA and 80%
acetonitrile as solvent B. Peptides were eluted using a 90 min
gradient up to 75% solvent B. The effluent of the HPLC column
enters a mixing tee, where it combines with matrix solution flowing
at 0.75 ul/min prior to spotting to a MALDI plate at the rate of 25
seconds per spot. The matrix solution was 4 mg/ml
alpha-cyano-4-hydroxycinnamic acid (Aldrich) in 70% acetonitrile
0.1% TFA containing 5 mM ammonium dihydrogen phosphate. The spots
were placed in a 25.times.40 array, where each HPLC gradient has
200 spots; thus, each Maldi plate can hold 5 reversed phase HPLC
gradients.
MALDI Tof Tof
[0713] The spotted plates contain 8 locations around the perimeter
of the plate where an external calibrant was placed. The external
calibrant was the "4700 mix" (Applied Biosystems) prepared as
directed. Upon insertion into the mass spectrometer, the laser was
aligned with the crosshairs of the sample viewer, and the plate was
aligned to the four corners of the 25.times.40 spot sample array.
The mass spectrometer was tuned for sensitivity and resolution
using the calibration spots, and then an external calibration was
obtained in MS and MSMS mode. Each HPLC gradient (200 spots) was
analyzed by acquiring an MS spectrum at each spot using 100 laser
shots randomly located at 10 positions within each sample spot.
Upon completion of the 200 MS spectra for each of the sample spots,
the data system determines the top 20 precursor ions per sample
spot (user selected variable) precursor ions for subsequent
acquisition of MS/MS spectra. Alternatively, specific precursor
masses can be acquired regardless of their presence or absence in
the MS spectra. Each MS/MS spectra was obtained from 2000 laser
shots from 20 random positions within each sample spot without
using collision gas. Upon completion, the MS/MS spectra can be
exported to the supplied database search engine (Mascot) for
peptide/protein identification. Analysis of the collected spectra
led to the identification of approximately 150 unique proteins from
plasma. When compared to the organ-specific proteins summarized in
Tables 1-32, it was shown that this dataset includes seven
glycosylated, tissue-specific proteins. These seven proteins are
listed in Table 34A and 34B.
TABLE-US-00003 TABLE 34A ORGAN-SPECIFIC GLYCOSYLATED PROTEINS
IDENTIFIED FROM A SAMPLE OF NORMAL HUMAN SERUM Speci- Gene
Accession Description Type Signature Class Count ficity CLU
NM_203339; Homo sapiens clusterin secr GATCCACCAGGCCT 4 80 0.64 SEQ
ID (complement lysis CAG; SEQ ID NO: 25362; inhibitor, SP-40, 40,
NO: 25654 NP_976084; sulfated glycoprotein 2, SEQ ID
testosterone-repressed NO: 25502 prostate message 2, apolipoprotein
J) (CLU), transcript variant 2, mRNA [Homo sapiens] C3 NM_000064;
Homo sapiens complement secr GATCTTGGGCCTTA 14 49 0.803 SEQ ID
component 3 (C3), mRNA GCA; SEQ ID NO: 5142; [Homo sapiens] NO:
6789 NP_000055; SEQ ID NO: 5976 IGJ NM_144646; Homo sapiens
immunoglobulin secr GATCACAGTTTGTTT 15 14 0.778 SEQ ID J
polypeptide, linker protein AC; SEQ ID NO: 8639; for immunoglobulin
alpha and NO: 11123 NP_653247; mu polypeptides (IGJ), mRNA SEQ ID
[Homo sapiens] NO: 9967 HRG NM_000412; Homo sapiens histidine-rich
secr GATCAAATGGAAAG 1 69 0.697 SEQ ID glycoprotein (HRG), mRNA GAG;
SEQ ID NO: 15453; [Homo sapiens] NO: 15808 NP_000403; SEQ ID NO:
15629 APOD NM_001647; Homo sapiens apolipoprotein D secr
GATCCAAGCAAAATC 14 42 1 SEQ ID (APOD), mRNA [Homo sapiens] CA; SEQ
ID NO: 15915; NO: 16713 NP_001638; SEQ ID NO: 16312 SERPING1
NM_000062; Homo sapiens serpin peptidase secr GATCAGGTTAGGGC 1 118
0.881 SEQ ID inhibitor, Glade G (C1 GAT; SEQ ID NO: 23547;
inhibitor), member 1, NO: 27220 NP_000053; (angioedema, hereditary)
SEQ ID (SERPING1), transcript variant NO: 23878 1, mRNA [Homo
sapiens] SERPING1 NM_001032295; Homo sapiens serpin peptidase secr
GATCAGGTTAGGGC 1 118 0.881 SEQ ID inhibitor, Glade G (C1 GAT; SEQ
ID NO: 23548; inhibitor), member 1, NO: 27220 NP_001027466;
(angioedema, hereditary) SEQ ID (SERPING1), transcript variant NO:
23879 2, mRNA [Homo sapiens] THBS1 NM_003246; Homo sapiens
thrombospondin 1 secr GATCACTTCTCCTTG 3 1002 0.809 SEQ ID (THBS1),
mRNA [Homo sapiens] GC; SEQ ID NO: 17925; NO: 18807 NP_003237; SEQ
ID NO: 18394
TABLE-US-00004 TABLE 34B ORGAN-SPECIFIC GLYCOSYLATED PROTEINS
IDENTIFIED FROM A SAMPLE OF NORMAL HUMAN SERUM expecta- peptide
tion peptide protein pep- Peptide Sequence Gene Accession Tissue
NXS/T length score score score tides (SEQ ID NO:) CLU NM_203339;
Spinal Yes 14 4.60E-08 93 108 2 LANLTQGEDQYYLR + SEQ ID Cord
Deamidation (N) NO: 25362; (SEQ ID NO: 50184) NP_976084; SEQ ID NO:
25502 C3 NM_000064; Brain Yes 28 8.90E-07 78 174 3
LVLSSEKTVLTPATNHMG SEQ ID Cere- NVTFTIPANR NO: 5142; bellum (+
Deamidation NP_000055; (N); Oxidation (M) SEQ ID (SEQ ID NO: 37801)
NO: 5976 IGJ NM_144646; Brain Yes 19 5.0E-06 68 68 1
IIVPLNNRENISDPTSPL SEQ ID Fetal R + Deamidation NO: 8639; (N) (SEQ
ID NP_653247; NO: 41099) SEQ ID NO: 9967 HRG NM_000412; Kidney Yes
19 1.40E-08 99 98 1 VIDFNCTTSSVSSALANT SEQ ID K + Deamidation NO:
15453; (N) (SEQ ID NP_000403; NO: 44534) SEQ ID NO: 15629 APOD
NM_001647; Lung Yes 26 3.10E-05 62 74 2 ADGTVNQIEGEATPVNLT SEQ ID
EPAKLEVK + NO: 15915; Deamidation (N) NP_001638; (SEQ ID SEQ ID NO:
45400) NO: 16312 SERPING1 NM_000062; Spleen Yes 21 1.20E-05 67 67 1
VLSNNSDANLELINTWVAK SEQ ID (SEQ ID NO: 49773) NO: 23547; NP_000053;
SEQ ID NO: 23878 SERPING1 NM_001032295; Spleen Yes 21 1.20E-05 67
67 1 VLSNNSDANLELINTWVAK SEQ ID (SEQ ID NO: 49773) NO: 23548;
NP_001027466; SEQ ID NO: 23879 THBS1 NM_003246; Mono- Yes 13
3.60E-05 65 65 1 VVNSTTGPGEHLR + SEQ ID cyte Deamidation (N) NO:
17925; (SEQ ID NO: 72689) NP_003237; SEQ ID NO: 18394
[0714] Thus, this example identifies numerous normal serum organ
specific proteins (Table 34A and 34B). These proteins are
diagnostically useful in a variety of settings as described herein,
for example, for defining a biological state of a subject and for
the diagnosis of specific diseases.
Example 3
Verification and Quantification of Serum Proteins Using
Enzyme-Linked Immunosorbant Assay (ELISA)
[0715] Blood serum tests to detect and monitor proteins were
developed using an enzyme-linked immunosorbent assay (ELISA). The
assay system utilized two antibodies directed against different
antigenic regions of the candidate protein. A monoclonal antibody
directed against a distinct antigenic determinant on the intact
candidate protein was used for solid phase immobilization on the
microtiter wells. A detection antibody conjugated to horseradish
peroxidase (HRP) or fluorescence tag recognized the candidate
protein within different region of the same protein. The candidate
protein reacted simultaneously with the two antibodies, resulting
in the protein being sandwiched between the solid phase and
detection antibody. The detection antibody was visualized by
colormetric fluorescence analysis.
[0716] In the case of peptides detected in blood, the specific
peptide was further enriched from peptide mixture isolated from
plasma using the physico-chemical properties of the peptide or
affinity reagents developed for the peptide. Protein concentrations
were estimated by an ELISA employing specific antibodies to capture
and detect the protein of interest in serum. Wells of 96-well
microtiter plates ("Maxisorp," Nunc, Roskilde, Denmark) were coated
with protein-specific antibodies and incubated overnight at
2-8.degree. C. Well surfaces were saturated with a solution of
irrelevant protein to prevent non-specific binding of subsequent
reactants and washed with Phosphate Buffered Saline (PBS), pH 7.2
with 0.05% Tween-20 (PBST) prior to use. Samples and reagents were
dispensed into the wells in the following sequence, each separated
by an incubation period and wash step: (1) serum samples and
protein concentration standards, (2) detection antibodies, (3)
horseradish peroxidase conjugate, and (4) peroxidase substrate. The
substrate reaction was stopped after a final incubation period with
the addition of acid to the wells, and the O.D.s were determined
with a microplate spectrophotometer. Sample concentrations were
then extrapolated from the dose-response curve.
Example 4
Identification of STEAP2 Protein Expression in Human Blood
Serum
A Comparison of Proteins in Normal Controls, Early Prostate Disease
and Late Stage Prostate Disease
[0717] Current methods for prostate cancer screening include cancer
screening with Prostate Specific Antigen (PSA). The PSA test is not
always predictive of prostate cancer due to individual patient risk
factors including ethnicity, family history, as well as the
patient's individual status and individual risk aversion to
complications from prostate cancer. Since the PSA test is not
entirely predictive, future prostate cancer screening will need to
incorporate new biomarkers to predict the risk of disease
(Thompson, et al., Surg Oncol Clin N Am. (2005) 14:747-60).
[0718] This example describes a multiparameter diagnostic
fingerprint using STEAP2, a multiple transmembrane protein of the
prostate as a biological marker. STEAP2 is the gene encoding a
Human, six transmembrane epithelial antigen of the prostate
(STEAP2). STEAP2 was previously shown to have prostate-specific
expression (Porkka, K P et al Lab Invest. (2002) 82:1573-1582;
Kormaz, K S, J. Biol. Chem. (2002) 27:36689-96). Further, as
outlined in Example 1 and shown in Table 21, STEAP2 was shown to be
a prostate-specific protein using the methods outlined herein.
[0719] Commercially available antibodies specific for numerous
proteins encoded by prostate-specific genes (see Table 21) were
used to determine which proteins would be useful in a
multiparameter diagnostic assay for prostate cancer. The antibody
available for STEAP2 (anti-STEAP2; Cat #A23080; Genway Biotech
Inc.) was shown to bind specifically to a fragment of STEAP2 from
human serum. In this example, TGM4 was used as a control. TGM4 is
the gene encoding Human prostate-specific transglutaminase (hTGP).
hTGP has prostate-specific expression (Dubbink, et al GENOMICS
(1998) 51:434-444). The antibody available for TGM4 (anti-TGM4; Cat
#G23082; Genway Biotech Inc.) was shown to specifically bind to
TGM4 from human serum.
[0720] Western blot analysis was used to measure serum protein
expression as follows: Serum was diluted (1:25) with sample buffer
(50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% sodium
dodecylsulphate, 0.1% bromophenol blue, 10% glycerol). Serum
proteins in 4 .mu.L of 4% serum solution were analyzed with
SDS-PAGE and transferred to a PVDF membrane (Hybond-P, Millipore,
Billerica, Mass.). The membrane was blocked with 1% non-fat dry
milk in TBS-T (25 mM Tris, pH 7.4, 125 mM NaCl, 0.1% Tween-20) for
1 hour at room temperature, followed by incubation with primary
antibodies against STEAP2 (1:5000) or TGM4 (1:5000) for 1 h at room
temperature (23.degree. C.). The membranes were washed 3 times with
TBS-T, and then incubated with horseradish peroxidase conjugated
anti-chicken antibodies (1:10,000) for 0.5 h. The immunoblot was
then washed five times with TBS-T and developed using enhanced
chemoluminescence following the manufacturer's recommendation
(Pierce) (see FIG. 2). The densities of the single bands
corresponding to STEAP2 and TGM4 were quantified using ImageJ
software (available at http colon double slash rsb dot info dot nih
dot gov/ij/). The results are summarized in Table 35. PSA scores
for each of the tested sera were obtained using a commercially
available ELISA kit. For these prostate cancer patients, the
majority of samples showed PSA levels above what is considered the
normal range (0-4 ng/mL). STEAP2 levels also appeared elevated in
many prostate cancer sera relative to sera from normal patients.
However, the combination of PSA and STEAP2 proved to be a better
predictor of prostate cancer than PSA levels alone, identifying
cancer in three samples with normal PSA levels (samples 7, 9, 10)
and in one sample with only slightly elevated PSA levels (sample
3).
TABLE-US-00005 TABLE 35 Relative density of STEAP2 and TGM4
determined using ImageJ software. Prostate Prostate Cancer Cancer
PSA TGM4 Sample ID Stage Progression (ng/mL) STEAP2 AU AU 1 Normal
Normal 1.6 0.09 8.4974 2 Normal Normal 1.8 1.95 9.5073 3 T1N0M0
Early PCa 5.56 11.95 8.8023 4 T2aN0MX Early PCa 4.06 6.68 9.3466 5
T1aNXM0 Early PCa 6.37 4.39 5.7375 6 T2aN0M0 Early PCa 12 12.23
1.3447 7 T2aN0M0 Early PCa 2.24 8.1 6.4363 8 Normal Normal 1.1 7.99
9.4223 9 T3N0Mx Late PCa 1.58 15.82 5.5885 10 T3aNXM0 Late PCa 2.84
9.5 7.3708 11 T3aNXM0 Late PCa 4.6 7.49 7.6887 12 T3bN0MX Late PCa
6.12 9.69 9.6478 13 Normal Normal 2.1 4.12 10.6099
[0721] In summary, the STEAP2 prostate-specific protein further
improved prostate cancer detection when used in combination with
PSA (see FIG. 2 and Table 35). Thus, using the methods described
herein, a multiparameter diagnostic panel was developed comprising
the STEAP2 prostate-specific protein and PSA.
Example 5
RNA Extraction from Organs Prior to Transcript Analysis
[0722] In further experiments, normal, healthy organ samples were
obtained. For each organ total RNA was isolated from each tissue as
follows: Tissue (0.1-0.2 mgs) from a specific organ was excised
from frozen tissue and placed in 4 mL of TRIZOL Reagent
(Invitrogen), (U.S. Pat. No. 5,346,994; Chomczynski et al.,
Analytical Biochemistry, (1987) 162:156). Each sample was quickly
homogenized for up to 90 seconds at room temperature. The
homogenate of tissue and TRIZOL Reagent was incubated for 5 min at
room temperature. Chloroform (800 uL) was added to each sample and
incubated for 5 min at room temperature. Each sample was
centrifuged at 12,000.times.g for 10 min at 4.degree. C. The
aqueous layer containing nucleic acids was collected and
transferred to fresh tubes. Nucleic acids were precipitated by the
addition of 2 mL of 2-propanol and incubated at room temperature
for 10 minutes. The precipitate was centrifuged at 12,000.times.g
for 10 minutes and nucleic acid pellets were collected. The
supernatants were decanted and pellets washed with 2 mL of 70%
ethanol and air dried. Pellets were suspended in 50-100 uL of RNase
free water. The nucleic acid concentration was calculated using
spectroscopy at wavelength (260 nm) and purity was determined by
calculating the ratio of absorbance at 260 nm and 280 nm in each
sample. Total RNA was analyzed by microcapillary electrophoresis
using a Bioanalyzer following the manufacturer's procedures
(Agilent 2100). Briefly, 1 uL of each RNA sample was diluted to 250
ng/uL in water was loaded onto a nano-CHIP and RNA profiles were
visualized using a standard protocol provided by the manufacturer.
The quality of the RNA was determined by examining the
electrophoresis peaks and by determining the relative ratio of the
28S and 18S ribosomal RNA. This ratio was used as a metric to
estimate the overall integrity of all other RNA species in the
sample. Samples where the electrophoresis peaks and the 28S:18S
ratio was greater than 1.5 were deemed acceptable for further
transcript analyses.
Example 6
Identification of Male and Female Organ-Specific Proteins
[0723] This example shows a further refinement and the
identification of organ-specific protein sets for prostate and
testes in the male and mammary gland and uterus in the female.
[0724] The normalized MPSS dataset described in Example 1 was
further refined to elicit nucleotide and protein sequences which
were specific to various male or female organs. To that end the
data from the thirty-two tissues described therein were delineated
further to identify sequences specific to male prostate (Table 36)
and testes (Table 37) and female mammary gland (Table 38) and
uterus (Table 39).
[0725] Refinement procedure to identify the organ-specific MPSS
sequences required examining the tissue-specific sequences of the
thirty-two tissues and subtracting the tissues specific for females
(in this case mammary gland and uterus) in order to fully examine
those sequences specific to male organs listed (particularly testes
and prostate). Similarly in order to examine organ-specific
sequences which relate specifically to female organs, subtraction
of the prostate and testes sequences from the thirty-two tissues
was performed.
[0726] Briefly, to identify nucleotide and protein sequences that
were specific to male organs (prostate and testis), MPSS data of
mammary gland and uterus were first removed from MPSS dataset of
the thirty-two tissues. MPSS data of the remaining thirty tissues
were then used to identify organ-specific MPSS sequences, using the
procedure described in Example 1. MPSS sequences that were specific
to prostate and testis were further annotated to their
corresponding nucleotide and protein sequences, as described in
Example 1. The obtained prostate-specific nucleotide and protein
sequences were summarized in Table 36 and the obtained
testis-specific nucleotide and protein sequences were summarized in
Table 37.
[0727] Similarly, to identify nucleotide and protein sequences that
were specific to female organs (mammary gland and uterus), MPSS
data of prostate and testis were first removed from MPSS dataset of
the thirty-two tissues that were described in Example 1. MPSS data
of the remaining thirty tissues were then used to identify
organ-specific MPSS sequences, as described in Example 1. MPSS
sequences that were specific to mammary gland and uterus were
further annotated to their corresponding nucleotide and protein
sequences, following the procedure described in Example 1. The
obtained mammary gland-specific nucleotide and protein sequences
were summarized in Table 38 and the obtained uterus-specific
proteins were summarized in Table 39.
Example 7
Identification of Potential Biomarkers for Prostate Cancer
[0728] This example shows the identification of protein sets that
are potential biomarkers for prostate cancer.
[0729] LNCaP cell line is a cellular model for early-stage prostate
cancer and CL1 cell line is a cellular model for late-stage
prostate cancer. Normalized MPSS dataset of LNCaP cells and CL1
cells were obtained for the purpose of identifying potential
biomarkers for prostate cancer. The new data were combined with the
normalized MPSS dataset of the thirty-two tissues that were
described in Example 1. Normal prostate-, LNCaP- and CL1-specific
nucleotide and protein sequences were identified from the combined
dataset and were potential biomarkers for prostate cancer.
[0730] More specifically, MPSS data of female organs (mammary gland
and uterus) were first removed from MPSS dataset of the thirty-two
tissues described in Example 1. MPSS data of the remaining thirty
tissues were then combined with normalized MPSS dataset of LNCaP
cells and CL1 cells. Following the procedure described in Example
1, the combined MPSS dataset of the thirty-two samples (the thirty
two tissues described in Example 1, minus mammary gland and uterus,
and plus LNCaP and CL1 cells) was used to identify nucleotide and
protein sequences that were specific to CL1 cells (Table 40), LNCaP
cells (Table 41) and normal prostate (Table 42). While normal
prostate-specific nucleotide and protein sequences are potential
biomarkers for the lack of prostate cancer, LNCaP-specific
nucleotide and protein sequences are potential biomarkers for
early-stage prostate cancer and CL1-specific nucleotide and protein
sequences are potential biomarkers for late-stage prostate cancer.
The obtained CL1-specific nucleotide and protein sequences were
summarized in Table 40, the obtained LNCaP-specific nucleotide and
protein sequences were summarized in Table 41, and the obtained
normal prostate-specific nucleotide and protein sequences were
summarized in Table 42.
Example 8
Collection of Organ-Specific Proteins that were Identified in Body
Fluids by Mass Spectrometry
[0731] Large amounts of mass spectrometry data on protein
identifications are accumulated very rapidly in the proteomics
field. Such data provide insightful information on the presence of
proteins in various biological specimens. This example shows the
collection of organ-specific proteins that have been identified by
mass spectrometry in body fluids such as serum, plasma and seminal
plasma.
[0732] Hundreds to thousands of proteins were identified in body
fluids such as serum, plasma and seminal plasma in several
proteomics studies and are stored in several proteomics databases.
The available mass spectrometry datasets on protein identifications
include: 1) human plasma proteins that were identified and managed
by the Human Proteome Organization (HUPO, Omenn, et al.,
Proteomics. (2005):3226-45), 2) peptides that were identified from
human plasma (PeptideAtlas, Deutsch et al., Proteomics
(2005):3497-500), 3) human plasma N-glycoproteins (Liu et al., J
Proteome Res (2005):2070-80), 4) human seminal plasma proteins
(Pilch B. and Mann M., Genome Biol (2006): R40), 5) N-glycopeptides
that were identified from various human specimens, and 6)
proprietary human serum protein databases. Proteins in the original
datasets were specified by their accession numbers in either
different protein sequence databases or different versions of the
databases. Thus proteins were not consistently annotated, which
made it impossible to directly combine proteins in different
datasets. To solve this problem, peptides that were directly
identified from mass spectrometry data were downloaded from
datasets 1)-6). These peptides were then assembled to proteins in
RefSeq database (ftp://ftp.ncbi.nih.gov/refseq/H_sapiens),
following a procedure described in the ProteinProphet algorithm
(Nesvizhskii et al., Anal Chem (2003): 4646-58). Hence we obtained
a list of proteins, and their belonging peptides, that were
identified in body fluids by mass spectrometry.
[0733] Proteins that were identified in body fluids by mass
spectrometry were then compared with organ-specific proteins
identified in Examples 1, 6, and 7. The overlap between proteins
identified in body fluids and organ-specific proteins listed in
Tables 1-32 was summarized in Table 43. The overlap between
proteins identified in body fluids and proteins specific to sex
organs (prostate, testis, mammary gland and uterus) that were
listed in Tables 36-39 was summarized in Table 44. The overlap
between proteins identified in body fluids and potential protein
biomarkers for prostate cancer that were listed in Tables 40-42 was
summarized in Table 45.
Example 9
Identification of Tissue Specific Genes and Proteins Using
Sequencing-by-Synthesis Analysis
[0734] This example shows the identification of organ-specific
protein sets from normal, healthy organs using
sequencing-by-synthesis (SBS).
[0735] SBS Dataset of Human Tissues
[0736] A total of 53 samples of human normal tissues and epithelial
cells were collected from different patients (see Table 46 for
details). These samples were different from the samples used to
generate the MPSS dataset described in Example 1. RNA molecules
were extracted from each sample using standard procedures (see
e.g., Example 5). Collected RNA samples were then sent to Solexa
(Hayward, Calif., USA, now part of Illumina, San Diego, Calif.,
USA) to measure the abundance of RNA molecules in each sample,
using their sequencing-by-synthesis (SBS) RNA analysis platform
(see e.g., Johnson D S, et al. (2007) Science 316(5830):1441-2; A.
Barski et al., 2007 Cell 129, 823-837; T. Mikkelsen et al., Nature.
2007 448(7153):553-60; G. Robertson et al., Nature Methods 2007
August; 4(8):651-7). Some samples were analyzed in duplicate (for
example, the two breast samples). As a result, a total of 64 SBS
datasets were received from Solexa (See Table 46).
[0737] Similar to MPSS data, each SBS dataset contains all
identified SBS tags, each tag comprised of a DNA sequence 20 bases
in length, their raw counts that qualify the abundance of the tags
in the sample, their annotations to the UniGene database, their
classification classes based on annotation quality, and
descriptions of the corresponding UniGene entries to which the tags
are annotated. This SBS dataset was then used to identify
organ-specific proteins as described below.
[0738] Assignment of Individual SBS Datasets to Organs
[0739] Some SBS datasets (such as HCC01_A and HCC01_B) were
generated from the same tissue. Some (such as HCC18 and HCC51) were
generated from tissues of the same organ but from different
patients. In addition, some samples were closely related to each
other (for example, hepatocytes and liver). To identify
organ-specific proteins, all SBS datasets were assigned to one of
the 25 organs listed in Table 46.
TABLE-US-00006 TABLE 46 List of all SBS datasets, the samples that
generated the datasets, and the organs to which the datasets were
assigned. Organ Sample Sex Patient ID SBS Dataset Adrenal Adrenal M
23209 HCC38 Gland Gland Artery Artery M 23060 HCC39 Bladder Bladder
F THB196 HCC11_A Bladder Bladder F THB196 HCC11_B Bladder Bladder M
23060 HCC10 Bladder Bladder M 21538 HCC42 Brain Brain F BR4-8L
HCC26 (Amygdala) Brain Brain F BR4-10L HCC27 (Nucleus Caudate)
Breast Breast F 108046 HCC01_A Breast Breast F 108046 HCC01_B
Breast Breast F 108046 HCC17_A Breast Breast F 108046 HCC17_B
Breast Breast F 108034 HCC19 Breast Breast F 108034 HCC02_A Breast
Breast F 108034 HCC02_B Cervix Cervix F 1-21 HCC05 Heart Heart F
19941 HCC51 Heart Heart M 23060 HCC18 Kidney Kidney F 301002 HCC53
Kidney Kidney M 301028 HCC52 Kidney Renal HCCHuECReCo Cortical
Epithelial Cells Kidney Renal HCCHuECRena Epithelial Cells Kidney
Renal HCCHuECRPT Proximal Tubule Epithelial Cells Liver Liver M
53891 HCC54 Liver Liver M 56310 HCC08 Liver Hepatocytes F HCCHuHep
Lung Lung F 301008 HCC56_A Lung Lung F 301008 HCC56_B Lung Lung F
301008 HCC56_C Lung Lung M AST6161 HCC55 Lymph Node Lymph Node F
20951 HCC46 Lymph Node Lymph Node F 19941 HCC57_A Lymph Node Lymph
Node F 19941 HCC57_B Lymph Node Lymph Node M THB196 HCC25
Lymphocytes Lymphocytes F NF11 + NF4 HCC14 (B) Lymphocytes
Lymphocytes M NMS10 HCC21 (B) Lymphocytes Lymphocytes F NF11 HCC15
(T) Monocytes Monocytes F NF11 HCC16 Monocytes Monocytes M NMS5
HCC20 Muscle Muscle M 54509 HCC58 (Skeletal) Muscle Muscle F 20951
HCC36 (Smooth) Ovary Ovary F 23011 HCC06 Pancreas Pancreas F 301002
HCC60 Pancreas Pancreas M 301001 HCC59 Pancreas Pancreatic F Islets
HCC40b Islet Cells Prostate Prostate M 23060 HCC03_A Prostate
Prostate M 23060 HCC03_B Prostate Prostate M 21538 HCC04 Prostate
Prostate M HCCHuECPros Epithelial Cells Skin Skin F 20951 HCC30
Skin Epidermal F HCCHuEK Keratinocytes Small Small F 301003 HCC62
Intestine Intestine Small Small M 21538 HCC31 Intestine Intestine
Spleen Spleen F 20951 HCC23 Spleen Spleen F 19941 HCC64 Spleen
Spleen M 21538 HCC50 Stomach Stomach F 19941 HCC65 Stomach Stomach
M 23060 HCC24 Stomach Stomach M 56310 HCC50A Testes Testes M 23060
HCC09 Thymus Thymus F 20951 HCC34 Thymus Thymus M 23060 HCC33
Trachea Trachea F 20951 HCC29 Uterus Uterus F 23011 HCC07
[0740] Identification of Organ-Specific SBS Tags
[0741] The methods for identifying organ-specific SBS tags were
similar to those for identifying organ-specific MPSS tags, as
described in Example 1. There were a few modifications to
accommodate difference in the two datasets. In particular, as
outlined further below, changes were made to account for having
multiple SBS datasets for some organs (see e.g., Table 46, multiple
samples for breast, spleen, lymph node, etc.).
[0742] One of the modifications was first to normalize raw counts
of SBS tags to transcription per million (tpm). In comparison, MPSS
data were already normalized to tpm. The methods for identifying
organ-specific SBS tags were as follows.
[0743] Assume the expression (in tpm) of a SBS sequence tag in the
64 SBS datasets was {X.sub.ij}, where i=1, 2, . . . , 25 represents
individual organs and j=1, 2, . . . , k.sub.i represents individual
datasets of the same organ. Apparently k.sub.i=1 if organ i had
only one dataset. For each organ, we first evaluated the following
three quantities:
[0744] (a) The highest expression of the tag in the organ,
i.e.,
X.sub.i.sup.m=max{X.sub.i1,X.sub.i2, . . . ,X.sub.ik.sub.i} (1)
[0745] (b) the averaged expression of the tag in the organ,
i.e.,
X i _ = 1 k i j = 1 k i X ij ; ( 2 ) ##EQU00015##
[0746] and (c) the corresponding standard deviation (SD) for
k.sub.i>1, i.e.,
s i = 1 k i - 1 j = 1 k i ( X ij - X i _ ) 2 . ( 3 )
##EQU00016##
[0747] If k.sub.i=1, one has X.sub.i.sup.m= X.sub.i=X.sub.i1 and
s.sub.i=0.
[0748] Assume organ m had the highest value of {X.sub.i.sup.m}
among all organs. We then applied three rules to determine whether
the tag was specific to organ m as follows:
[0749] i) The highest expression of the tag in organ m was above a
minimal, estimated noise level, i.e.,
X.sub.m.sup.m.gtoreq.5. (4)
[0750] ii) The highest expression of the tag in organ m was well
above the averaged expression of the tag in all other tissues. More
specifically, we first calculated the mean averaged expression of
the tag in the other 24 organs (except organ m) as
X _ = 1 N i .noteq. m X i _ , ( 5 ) ##EQU00017##
[0751] the associated standard error as
.sigma. X _ = 1 N i .noteq. m s i 2 , ( 6 ) ##EQU00018##
[0752] and the corresponding SD as
s = 1 N - 1 i .noteq. m ( X _ i - X _ ) 2 + 1 N i .noteq. m s i 2 ,
( 7 ) ##EQU00019##
[0753] where N=24.
[0754] We then evaluated the significance that the expression of
the tag in organ m was above the expression of the tag in other
organs as
p dis = 1 2 erfc ( X m m - X _ 2 ( s 2 + .sigma. X 2 ) ) . ( 8 )
##EQU00020##
[0755] For the tag to be specific to organ m, we required that
p.sub.dis.ltoreq.10.sup.-3. (9)
[0756] iii) The specificity f of the tag in organ m had to be well
above a pre-selected cutoff value f.sub.0. More precisely, we
defined the specificity of the tag in tissue m as
f = X m m X m m + i .noteq. m X _ i , ( 10 ) ##EQU00021##
[0757] and evaluated the associated SD as
.sigma. f = f 2 X m m i .noteq. m s i 2 . ( 11 ) ##EQU00022##
[0758] The significance that f was above f.sub.0 is then given
by
p spc = 1 2 erfc ( f - f 0 2 .sigma. f ) . ( 12 ) ##EQU00023##
[0759] For the tag to be specific to organ m, we selected
f.sub.0=0.5 (13)
[0760] and required that
p.sub.spc.ltoreq.0.1. (14)
[0761] A tag was identified as specific to organ m if its
expression satisfied the three conditions in Eqs. (4), (9) and
(14).
[0762] Lists of Organ-Specific Proteins
[0763] The mapping of organ-specific SBS tags to organ-specific
proteins was the same as that for the MPSS data as described in
Example 1.
[0764] All organ-specific proteins identified from the SBS data
were listed in Tables 47-71.
[0765] All proteins discovered from SBS data as specific to male or
female sex organs were listed in Tables 72-77. The methods for this
analysis are as described in Example 6.
[0766] All organ-specific proteins discovered from SBS data and
previously identified by mass spectrometry were listed in Table 78.
The methods for this analysis are as described in Example 8.
[0767] All proteins discovered from SBS data as specific to male or
female sex organs and previously identified by mass spectrometry
were listed in Table 79. The methods for this analysis are as
described in Example 8.
[0768] Information on each protein listed in Tables 47-79 includes
gene name, gene accession number, protein accession number, summary
description of the protein, predicted protein localization (as
described in Example 1), corresponding MPSS tag, the class of the
MPSS tag, the expression of the tag in the particular tissue
(count), the specificity of the tag in the particular tissue (as
described above), and the number of peptides identified on the
protein by HUPO (as described further below). Those proteins
identified by MPSS and by SBS are noted by "&". The amino acid
sequences corresponding to the organ-specific proteins and the
polynucleotides encoding the proteins are provided in the sequence
listing. Sequences corresponding to the MPSS tags as shown in
Tables 47-79 are also provided. Sequences corresponding to peptides
previously identified by mass spectrometry that map to
organ-specific proteins described in Tables 78 and 79 are shown in
those Tables and are provided in the sequence listing.
[0769] In summary, the experiments described in this Example
identified organ-specific protein sets as set forth in Tables
47-79. These proteins are diagnostically and therapeutically useful
in a variety of settings as described herein, for example, for
defining a biological state of a subject and for the diagnosis of
specific diseases. These proteins and detection reagents thereto
can be used in accurate assays, panels, arrays and methods to
measure health, detect disease and to monitor treatment.
[0770] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0771] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140106981A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140106981A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References