U.S. patent application number 15/019954 was filed with the patent office on 2016-06-02 for complement assays and uses thereof.
The applicant listed for this patent is Apellis Pharmaceuticals, Inc.. Invention is credited to Pascal Deschatelets, Cedric Francois, Paul Olson, Zhouning Zhang.
Application Number | 20160153985 15/019954 |
Document ID | / |
Family ID | 43126807 |
Filed Date | 2016-06-02 |
United States Patent
Application |
20160153985 |
Kind Code |
A1 |
Zhang; Zhouning ; et
al. |
June 2, 2016 |
COMPLEMENT ASSAYS AND USES THEREOF
Abstract
The present invention provides methods for assessing complement
activation and methods for assessing the ability of an agent or
condition of interest to modulate complement activation. The
present invention provides methods for assessing whether a subject
has or is at increased risk of developing a complement-mediated
disorder, e.g., age-related macular degeneration (AMD). Also
provided are kits containing materials useful for performing the
methods.
Inventors: |
Zhang; Zhouning;
(Louisville, KY) ; Francois; Cedric; (Prospect,
KY) ; Deschatelets; Pascal; (Prospect, KY) ;
Olson; Paul; (St. Louis, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Apellis Pharmaceuticals, Inc. |
Crestwood |
KY |
US |
|
|
Family ID: |
43126807 |
Appl. No.: |
15/019954 |
Filed: |
February 9, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13321522 |
Feb 9, 2012 |
9291622 |
|
|
PCT/US10/35871 |
May 21, 2010 |
|
|
|
15019954 |
|
|
|
|
61230645 |
Jul 31, 2009 |
|
|
|
61180273 |
May 21, 2009 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/7.92;
435/7.93; 435/7.94; 435/7.95; 436/501; 506/18 |
Current CPC
Class: |
G01N 33/6893 20130101;
G01N 2800/164 20130101; C07K 16/18 20130101; G01N 33/566 20130101;
G01N 2800/7042 20130101; G01N 2333/4716 20130101; G01N 33/564
20130101; G01N 2800/50 20130101; G01N 2800/52 20130101; G01N
2800/16 20130101 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Claims
1. A method of assessing complement activation in a sample
comprising determining the amount of iC3b, the ratio of iC3b to
intact C3, or the fraction of C3 that is intact in the sample.
2. The method of claim 1, wherein the method comprises determining
the ratio of iC3b to intact C3 in the sample.
3. The method of claim 1, wherein the sample comprises whole blood,
plasma, or serum.
4. The method of claim 1, wherein the sample comprises aqueous
humor or vitreous humor.
5. The method of claim 1, wherein the sample was obtained from a
human subject.
6. The method of claim 1, wherein the sample was obtained from a
subject having or suspected of having a complement deficiency or
complement-mediated disease or condition.
7. The method of claim 1, wherein the sample was obtained from a
subject suffering from AMD.
8. The method of claim 1, wherein the sample was obtained from a
subject who has recently suffered severe injury, stroke, asthma
exacerbation, cardiac arrest, or heart attack.
9. The method of claim 1, wherein the sample was obtained from a
subject who has been treated with a complement inhibitor.
10. The method of claim 1, wherein the sample comprises an agent
being assessed for its ability to modulate complement
activation.
11. The method of claim 1, comprising steps of: (a) determining the
concentration of intact C3 present in the sample; (b) determining
the concentration of iC3b present in the sample; (c) calculating
the ratio of iC3b to intact C3 in the sample using the results of
steps (a) and (b).
12. The method of claim 1, comprising steps of: (a) determining the
concentration of intact C3 present in the sample; (b) determining
the concentration of total C3 present in the sample; and (c)
calculating the fraction of C3 that is intact in the sample using
the results of steps (a) and (b).
13. The method of claim 1, comprising steps of: (a) capturing
intact C3 present in one or more sub-samples of the sample using a
first capture agent that binds to intact C3; (b) quantifying intact
C3 captured in step (a); (c) capturing iC3b present in one or more
sub-samples of the sample using a second capture agent that binds
to a neoepitope of iC3b; (d) quantifying iC3b captured in step (c);
and (e) calculating the ratio of iC3b to intact C3 in the sample
using the results of steps (b) and (d).
14. The method of claim 1, comprising steps of: (a) capturing
intact C3 present in one or more sub-samples of the sample using a
first capture agent that binds to intact C3; (b) quantifying intact
C3 captured in step (a); (c) capturing total C3 present in one or
more sub-samples of the sample using a second capture agent that
binds to total C3; (d) quantifying total C3 captured in step (c);
and (e) calculating the fraction of C3 that is intact in the sample
using the results of steps (b) and (d).
15. The method of claim 13 or 14, wherein the first capture agent,
the second capture agent, or both, are each immobilized on a
support.
16. The method of claim 15, wherein the support comprises a
microwell plate.
17. The method of claim 15, wherein the support comprises a
plurality of particles.
18. The method of claim 13 or 14, wherein the capture agent of step
(a), step (c), or both comprises an antibody.
19. The method of claim 13 or 14, wherein the capture agent of step
(a) comprises an antibody that binds to C3a.
20. The method of claim 19, wherein the antibody is monoclonal.
21. The method of claim 13, wherein the capture agent of step (c)
comprises an antibody that binds to a neoepitope of iC3b.
22. The method of claim 21, wherein the antibody is monoclonal.
23. The method of claim 14, wherein the capture agent of step (a)
comprises an antibody that binds to C3d.
24. The method of claim 14, wherein step (b) comprises detecting
intact C3 using a detection agent that specifically binds to
C3a.
25. The method of claim 13, wherein step (a) comprises capturing
intact C3 using a monoclonal antibody that binds to C3a, and step
(b) comprises detecting intact C3 using a polyclonal antibody that
binds to C3.
26. The method of claim 13, wherein step (c) comprises capturing
iC3b using a monoclonal antibody that binds to a neoepitope of
iC3b, and step (b) comprises detecting iC3b using a polyclonal
antibody that binds to C3.
27. The method of claim 14, wherein the capture agent that binds to
total C3 is a polyclonal antibody that binds to C3d and the
specific binding agent for detecting intact C3 is a monoclonal
antibody that binds to C3a.
28. The method of claim 14, wherein step (d) comprises detecting
total C3 using a detection agent that binds to total C3.
29. The method of claim 28, wherein the detection agent for
detecting total C3 comprises a monoclonal antibody that binds to
C3d.
30. The method of claim 28, wherein the detection agent for
detecting total C3 comprises a polyclonal antibody that binds to
C3.
31. The method of claim 1, wherein the method comprises determining
the ratio of iC3b to intact C3, further comprising determining the
concentration of total C3 in the sample.
32. A method of assessing complement activation in a subject
comprising steps of: (a) providing a biological sample obtained
from the subject; and (b) assessing complement activation in the
sample according to the method of claim 1.
33. The method of claim 32, further comprising (c) using the result
of step (b) to provide diagnostic, prognostic, or treatment-related
information regarding the subject.
34. A method of assessing complement activation in a sample
comprising (a) capturing and detecting intact C3, wherein intact C3
is captured using an antibody that binds to C3a but does not bind
to other C3 fragments; and (b) capturing and detecting iC3b,
wherein iC3b is captured using an antibody that binds to a
neoepitope of iC3b, and wherein the ratio of iC3b to intact C3
serves as an indicator of the extent of complement activation, with
a higher ratio indicating a greater extent of complement
activation.
35. The method of claim 34, wherein the antibody that binds to C3a
is a monoclonal antibody.
36. The method of claim 34, wherein the antibody that binds to iC3b
is a monoclonal antibody.
37. The method of claim 34, wherein intact C3 and iC3b are detected
using the same detection agent.
38. The method of claim 33, wherein intact C3 and iC3b are detected
using a polyclonal antibody.
39. The method of claim 38, wherein the polyclonal antibody binds
to C3.
40. The method of claim 34, wherein intact C3 is captured using an
antibody that binds to C3a and is detected using an antibody that
binds to C3.
41. The method of claim 34, wherein intact C3 is captured using a
monoclonal antibody that binds to C3a and is detected using a
polyclonal antibody that binds to C3.
42. The method of claim 34, wherein intact C3 is captured using a
monoclonal antibody that binds to C3a and is detected using a
polyclonal antibody that binds to C3d.
43. The method of claim 34, wherein iC3b is captured using a
monoclonal antibody that binds to a neoepitope of iC3b and is
detected using a polyclonal antibody that binds to C3.
44. The method of claim 34, wherein total C3 is captured using a
polyclonal antibody that binds to C3d and is detected using a
monoclonal antibody that binds to C3d.
45. The method of claim 34, wherein intact C3 is captured using a
monoclonal antibody that binds to C3a and is detected using a
polyclonal antibody that binds to C3, and iC3b is captured using a
monoclonal antibody that binds to a neoepitope of iC3b and is
detected using a polyclonal antibody that binds to C3.
46. The method of claim 45, wherein the same polyclonal antibody is
used to detect intact C3 and to detect iC3b.
47. The method of claim 34, wherein the antibody of step (a) and
the antibody of step (b) are attached to supports.
48. The method of claim 34, wherein the sample comprises serum or
plasma.
49. The method of claim 34, wherein the sample comprises aqueous or
vitreous humor.
50. The method of claim 34, wherein the sample was obtained from a
human subject.
51. The method of claim 34, wherein the sample was obtained from a
subject having or suspected of having a complement deficiency or
complement-mediated disease or condition.
52. The method of claim 34, wherein the sample was obtained from a
subject suffering from AMD.
53. The method of claim 34, wherein the sample was obtained from a
subject who has recently suffered severe injury.
54. The method of claim 34, wherein the sample was obtained from a
subject being treated with a complement inhibitor.
55. The method of claim 34, wherein the sample comprises an agent
being assessed for its ability to modulate complement
activation.
56. A method of assessing complement activation in a subject
comprising steps of: (a) providing a sample obtained from the
subject; and (b) assessing complement activation in the sample
according to the method of claim 34.
57. The method of claim 56, further comprising using the result of
step (b) to provide diagnostic, prognostic, or treatment-related
information regarding the subject.
58. A kit comprising: (a) a capture agent that binds to intact C3
and does not bind to C3b or iC3b; (b) a capture agent that binds to
a neoepitope of iC3b; and (c) a detection agent that binds to
intact C3 and (d) a detection agent that binds to iC3b.
59. The kit of claim 58, wherein the capture agent of (a) comprises
a monoclonal antibody that binds to C3a.
60. The kit of claim 58, wherein the capture agent of (b) comprises
a monoclonal antibody that binds to a neoepitope of iC3b.
61. The kit of claim 58, wherein the detection agent of (c) and the
detection agent of (d) are the same.
62. The kit of claim 58, wherein the detection agent that binds to
intact C3 comprises an antibody that binds to C3.
63. The kit of claim 62, wherein the antibody that binds to C3 is a
polyclonal antibody.
64. The kit of claim 58, wherein the kit comprises (i) a polyclonal
antibody that binds to C3; and (ii) a monoclonal antibody that
binds to C3a.
65. The kit of claim 58, wherein the kit comprises (i) a monoclonal
antibody that binds to a neoepitope of iC3b; and (ii) a monoclonal
antibody that binds to C3a.
66. The kit of claim 58, wherein the kit comprises (i) a polyclonal
antibody that binds to C3; (ii) a monoclonal antibody that binds to
a neoepitope of iC3b; (iii) and a monoclonal antibody that binds to
C3a.
67. The kit of claim 58, further comprising at least one item
selected from the group consisting of: instructions for use of the
kit to assess complement activation; one or more native complement
components or cleavage products; human serum complement, optionally
characterized for levels of one or more complement components or
cleavage products; serum depleted of one or more complement
components; a detectably labeled secondary antibody; an enzyme
substrate; a buffer; and a support.
68. The kit of claim 67, wherein the kit comprises one or more
proteins selected from the group consisting of: C3 protein; C3d
polypeptide; C3b polypeptide, iC3b polypeptide.
69. The kit of claim 67, wherein the detectably labeled secondary
antibody is labeled with an enzyme.
70. A method for assessing whether a subject has, or is at
increased risk of developing, age-related macular degeneration
(AMD), or is at increased risk of progressing from early AMD to
more advanced AMD, comprising detecting intact C3 in a sample
comprising serum, plasma, or blood from the subject, wherein a
level of intact C3 that is greater than a control level indicates
that the individual has or is at increased risk of developing AMD,
or is at increased risk of progressing from early AMD to more
advanced AMD.
71. The method of claim 70, wherein the control level is a level
found in a control population composed of individuals not having
AMD.
72. The method of claim 71, wherein the control population is
composed of individuals at least 60 years of age.
73. The method of claim 70, wherein the method comprises detecting
intact C3 using an ELISA assay.
74. The method of claim 70, wherein the method comprises capturing
intact C3 using a monoclonal antibody that binds to C3a and
detecting captured intact C3 using a polyclonal antibody that binds
to C3.
75. The method of claim 70, wherein the method comprises capturing
intact C3 using a monoclonal antibody that binds to C3a and
detecting intact C3 using a polyclonal antibody that binds to C3d
and does not significantly recognize C3 epitopes outside C3d.
76. The method of claim 74 or 75, wherein the monoclonal antibody
is mouse anti-human C3a/C3 antibody #HM2075 (Cell Sciences) or an
antibody that binds to the same epitope.
77. The method of claim 70, wherein the subject has not been
diagnosed with AMD.
78. The method of claim 70, wherein the individual has early AMD,
and wherein a level of intact C3 that is greater than a control
level indicates that the individual is at increased risk of
progressing from early AMD to more advanced AMD.
79. The method of claim 70, wherein the individual has early AMD,
and wherein a level of intact C3 that is greater than a control
level indicates that the individual is progressing to more advanced
AMD.
80. A method for assessing whether a subject has or is at increased
risk of developing age-related macular degeneration (AMD)
comprising determining the level of one or more proteins in a
sample comprising serum, plasma, or blood from the subject using a
monoclonal antibody that binds to C3a/C3, wherein a level of said
one or more proteins that is greater than a control level indicates
that the individual has or is at increased risk of developing
AMD.
81. The method of claim 80, wherein the method comprises capturing
said one or more proteins using the monoclonal antibody that binds
to C3a and detecting captured intact C3 using a polyclonal antibody
that binds to C3.
82. The method of claim 80, wherein the method comprises capturing
said one or more proteins using the monoclonal antibody that binds
to C3a and detecting captured intact C3 using a polyclonal antibody
that binds to C3d and does not significantly recognize C3 epitopes
outside C3d.
83. The method of claim 80, wherein the monoclonal antibody is
mouse anti-human C3a/C3 antibody #HM2075 (Cell Sciences) or an
antibody that binds to the same epitope.
84. The method of claim 80, wherein the control level is a level
found in a control population composed of individuals not having
AMD.
85. The method of claim 84, wherein the control population is
composed of individuals at least 60 years of age.
86. The method of claim 80, wherein the subject has not been
diagnosed with AMD.
87. A kit comprising: (a) a capture agent that binds to intact C3
and does not bind to C3b or iC3b; and (b) a detection agent that
binds to intact C3.
88. The kit of claim 87, wherein the capture agent comprises a
monoclonal antibody that binds to C3a.
89. The kit of claim 87, wherein the detection agent comprises a
polyclonal antibody that binds to C3.
90. The kit of claim 87, further comprising at least one item
selected from the group consisting of: instructions for use of the
kit to assess levels of intact C3; intact C3 protein; a detectably
labeled secondary antibody; an enzyme substrate; a buffer; and a
support.
91. A method for measuring intact C3 in a sample comprising serum,
plasma, blood, or another body fluid obtained from a subject,
wherein the method comprises capturing intact C3 using a monoclonal
antibody that binds to C3a and detecting captured intact C3 using a
polyclonal antibody that binds to C3.
92. The method of claim 91, further comprising comparing the level
of intact C3 in the sample with a control level.
93. The method of claim 91, wherein the method comprises detecting
intact C3 using an ELISA assay.
94. The method of claim 91, wherein the method comprises capturing
intact C3 using a monoclonal antibody that binds to C3a and
detecting intact C3 using a polyclonal antibody that binds to C3d
and does not significantly recognize C3 epitopes outside C3d.
95. The method of any of claims 91-94, wherein the monoclonal
antibody is mouse anti-human C3a/C3 antibody #HM2075 (Cell
Sciences) or an antibody that binds to the same epitope.
Description
RELATED APPLICATIONS
[0001] The present application claims priority to, and benefit of,
U.S. provisional patent application nos. U.S. Ser. No. 61/180,273
(filed May 21, 2009) and U.S. Ser. No. 61/230,645 (filed Jul. 31,
2009), the entire contents of each of which are herein incorporated
by reference.
BACKGROUND OF THE INVENTION
[0002] The complement system comprises more than 30 serum and
cellular proteins and has important roles in innate and adaptive
immunity (Walport, M., N Engl J Med.; 344(14):1058-66, 2001).
Complement activation can occur via three major pathways: the
classical, alternative, and lectin pathways. The classical pathway
is typically triggered by binding of a complex of antigen and IgM
or IgG antibody to complement component C1. Activated C1 cleaves
components C4 and C2 to produce C4a and C4b, in addition to C2a and
C2b. C4b and C2a combine to form the classical pathway (CP) C3
convertase, which cleaves C3 to form C3a and C3b. Binding of C3b to
C3 convertase produces a C5 convertase. The alternative pathway
(AP) is typically activated by targets such as microbial surfaces
and various complex polysaccharides and other materials. This
pathway can be initiated by spontaneous cleavage of the thioester
bond in C3 to form C3(H.sub.2O). C3(H.sub.2O) binds factor B, which
allows factor D to cleave factor B to Ba and Bb. Bb remains
associated with C3(H.sub.2O) to form the C3(H.sub.2O)Bb complex,
which acts as a C3 convertase and cleaves C3, resulting in C3a and
C3b. C3b formed either via this process or via the classical or
lectin pathways binds to targets, e.g., on cell surfaces, and forms
a complex with factor B, which is later cleaved by factor D to form
Bb, resulting in a C3 convertase. This alternative pathway
convertase is also termed C3bBb. Binding of another molecule of C3b
to the C3 convertase produces a C5 convertase. The lectin
complement pathway is initiated by binding of mannose-binding
lectin (MBL) and MBL-associated serine protease (MASP) to
carbohydrates. The MB1-1 gene (known as LMAN-1 in humans) encodes a
type I integral membrane protein localized in the intermediate
region between the endoplasmic reticulum and the Golgi. The MBL-2
gene encodes the soluble mannose-binding protein found in serum. In
the human lectin pathway, MASP-1 and MASP-2 are involved in
proteolysis of C4 and C2, leading to a C3 convertase, which leads
to production of a C5 convertase as described above for the
classical pathway.
[0003] C5 convertases generated via any of the pathways cleave C5
to produce C5a and C5b. C5b then binds to C6, C7, and C8 to form
C5b-8, which catalyzes polymerization of C9 to form the C5b-9
membrane attack complex (MAC). The MAC inserts itself into target
cell membranes and causes cell lysis. Sub-lytic amounts of MAC on
the membrane of cells may affect cell function in a variety of
ways. The small cleavage products C3a, C4a, and C5a are
anaphylotoxins and mediate multiple reactions in the acute
inflammatory response. C3a and C5a are also chemotactic factors
that attract immune system cells such as neutrophils. All three
anaphylatoxins contain carboxyl-terminal arginine residues that are
rapidly removed by carboxypeptidases in whole blood or serum. The
desarginine derivatives of C3a and C4a exhibit minimal binding to
the C3a receptor. In the case of C5a, the biologic activity and
decreases by more than an order of magnitude.
[0004] Deficiencies and functional disorders in complement
components or regulatory proteins are responsible for a number of
disorders. It is also increasingly recognized that excessive or
inappropriate complement activation plays an important pathogenic
role in a wide variety of diseases and clinical conditions. A
variety of assays have been developed for studying the complement
system, diagnosing complement deficiencies and complement-mediated
diseases, and developing agents to modulate the complement system.
Hemolysis-based techniques may be used to measure the functional
activity of the entire complement cascade. Classical or alternative
complement pathway activation may be assessed by measuring
complement-mediated hemolysis of erythrocytes (e.g.,
antibody-sensitized or unsensitized sheep or rabbit erythrocytes)
by human serum or by a set of complement components. For example,
to measure the functional capacity of the classical pathway, sheep
red blood cells coated with hemolysin (rabbit IgG to sheep red
blood cells) are typically used as target cells (sensitized cells).
These Ag-Ab complexes activate the classical pathway and result in
lysis of target cells when the complement components are functional
and present in sufficient concentration. To determine the
functional capacity of the alternative pathway, rabbit red blood
cells are typically used as the target cell.
[0005] A variety of immunological techniques have been developed to
measure the concentration of a particular complement component or
cleavage product. Antibodies have been raised against different
epitopes of various (human) complement components and their major
cleavage products. Radioimmunoassays, radial diffusion assays, and
enzyme-linked immunosorbent assays (ELISAs) have been developed to
detect these molecules. Descriptions of various methods that have
been employed in efforts to measure complement activation are found
in Wurzer, R., "Immunochemical Measurement of Complement Components
and Activation Products", pp. 103-112, in Complement Methods and
Protocols (Methods in Molecular Biology) Humana Press; 1 edition,
2000, in the review article by Mollnes, T., et al., "Complement
analysis in the 21.sup.st century", Mol. Immunol., 44: 3838-3849,
2007, and references therein, all of which are incorporated herein
by reference.
SUMMARY OF THE INVENTION
[0006] The present invention provides methods for assessing the
level of intact complement component C3 in a sample. The invention
also provides methods for assessing the level of iC3b in a sample
and methods for assessing the amount of total C3 in a sample. The
methods may be used individually, or two or more methods may be
used together, as described further below. In some embodiments the
sample comprises blood, plasma, or serum. In some embodiments the
sample comprises aqueous humor or vitreous humor. In some
embodiments the sample was obtained from a human subject. In some
embodiments the sample was obtained from a subject suffering from
AMD. In some embodiments the subject has not been previously
diagnosed as having AMD. In some embodiments the subject has been
previously diagnosed as having dry AMD and not wet AMD. In some
embodiments the subject has been previously diagnosed as having
early AMD and not geographic atrophy (GA). In some embodiments the
subject has been previously diagnosed as having GA in one eye and
not the other eye. In some embodiments the subject has been
previously diagnosed as having GA not involving the fovea.
[0007] The invention further provides a method for assessing
whether a subject has, or is at increased risk of developing,
age-related macular degeneration (AMD), or is at increased risk of
progressing from early AMD to more advanced AMD, comprising
detecting intact C3 in a sample comprising serum, plasma, or blood
from the subject, wherein a level of intact C3 that is greater than
a control level indicates that the individual has or is at
increased risk of developing AMD, or is at increased risk of
progressing from early AMD to more advanced AMD.
[0008] The invention further provides a method for assessing
whether a subject has or is at increased risk of developing
age-related macular degeneration (AMD) comprising determining the
level of one or more proteins in a sample comprising serum, plasma,
or blood from the subject using a monoclonal antibody that binds to
C3a, wherein a level of said one or more proteins that is greater
than a control level indicates that the individual has or is at
increased risk of developing AMD.
[0009] In some aspects, the invention provides methods of assessing
complement activation. In some aspects, the invention provides a
method of assessing complement activation in a sample comprising
determining the amount of iC3b, the ratio of iC3b to intact C3, or
the fraction of C3 that is intact in the sample. In some
embodiments the method comprises determining the ratio of iC3b to
intact C3 in the sample. In some embodiments the sample comprises
whole blood, plasma, or serum. In some embodiments the sample
comprises aqueous humor or vitreous humor. In some embodiments the
sample was obtained from a human subject. In some embodiments the
sample was obtained from a subject having or suspected of having a
complement deficiency or complement-mediated disease or condition.
In some embodiments the sample was obtained from a subject
suffering from AMD. In some embodiments the subject suffers from
early AMD. In some embodiments the subject has a genotype
associated with increased risk of developing AMD. In some
embodiments the sample was obtained from a subject who has recently
suffered severe injury, stroke, asthma exacerbation, cardiac
arrest, or heart attack. In some embodiments the sample was
obtained from a subject who has been treated with a complement
inhibitor. In some embodiments the sample comprises an agent being
assessed for its ability to modulate complement activation. In some
embodiments the method comprises steps of: (a) determining the
concentration of intact C3 present in the sample; (b) determining
the concentration of iC3b present in the sample; and (c)
calculating the ratio of iC3b to intact C3 in the sample using the
results of steps (a) and (b). In some embodiments the method
comprises steps of: (a) determining the concentration of intact C3
present in the sample; (b) determining the concentration of total
C3 present in the sample; and (c) calculating the fraction of C3
that is intact in the sample using the results of steps (a) and
(b). In some embodiments the method comprises steps of: (a)
capturing intact C3 present in one or more sub-samples of the
sample using a first capture agent that binds to intact C3; (b)
quantifying intact C3 captured in step (a); (c) capturing iC3b
present in one or more sub-samples of the sample using a second
capture agent that binds to a neoepitope of iC3b; (d) quantifying
iC3b captured in step (c); and (e) calculating the ratio of iC3b to
intact C3 in the sample using the results of steps (b) and (d). In
some embodiments the method comprises steps of: (a) capturing
intact C3 present in one or more sub-samples of the sample using a
first capture agent that binds to intact C3; (b) quantifying intact
C3 captured in step (a); (c) capturing total C3 present in one or
more sub-samples of the sample using a second capture agent that
binds to total C3; (d) quantifying total C3 captured in step (c);
and (e) calculating the fraction of C3 that is intact in the sample
using the results of steps (b) and (d). In some embodiments of a
method that employs a capture agent, the first capture agent, the
second capture agent, or both, are each immobilized on a support.
In some embodiments the support comprises a microwell plate. In
some embodiments the support comprises a plurality of particles. In
some embodiments of a method that employs a capture agent, e.g.,
the capture agent of step (a), step (c), or both comprises an
antibody. In some embodiments a capture agent that binds to intact
C3 comprises an antibody that binds to C3a. Such an antibody is
sometimes referred to herein as binding to C3a/C3, indicating that
the antibody binds to the C3a portion of C3 and recognizes its
epitope when present in intact C3. In some embodiments the antibody
is monoclonal. In some embodiments a capture agent that binds to a
neoepitope of iC3b comprises an antibody that binds to a neoepitope
of iC3b. In some embodiments the antibody is monoclonal. In some
embodiments a capture agent that binds to total C3 comprises an
antibody that binds to C3d. In some embodiments intact C3 is
detected and optionally quantified using a detection agent that
specifically binds to C3a. In some embodiments an inventive method
comprises capturing intact C3 using a monoclonal antibody that
binds to C3a, and detecting intact C3 using a polyclonal antibody
that binds to C3. In some embodiments an inventive method comprises
capturing iC3b using a monoclonal antibody that binds to a
neoepitope of iC3b and detecting iC3b using a polyclonal antibody
that binds to C3. In some embodiments a capture agent that binds to
total C3 is a polyclonal antibody that binds to C3d and a specific
binding agent for detecting intact C3 is a monoclonal antibody that
binds to C3a. In some embodiments the method comprises detecting
total C3 using a detection agent that binds to total C3. In some
embodiments the detection agent for detecting total C3 comprises a
monoclonal antibody that binds to C3d. In some embodiments the
method the detection agent for detecting total C3 comprises a
polyclonal antibody that binds to C3. In some embodiments the
method comprises determining the ratio of iC3b to intact C3,
further comprising determining the concentration of total C3 in the
sample.
[0010] In another aspect, the invention provides a method of
assessing complement activation in a sample comprising: (a)
capturing and detecting intact C3, wherein intact C3 is captured
using an antibody that binds to C3a but does not bind to other C3
fragments; and (b) capturing and detecting iC3b, wherein iC3b is
captured using an antibody that binds to a neoepitope of iC3b, and
wherein the ratio of iC3b to intact C3 serves as an indicator of
the extent of complement activation, with a higher ratio indicating
a greater extent of complement activation. In some embodiments the
antibody that binds to C3a is a monoclonal antibody. In some
embodiments the antibody that binds to iC3b is a monoclonal
antibody. In some embodiments the detection agent used to detect
intact C3 is the same as the detection agent used to detect iC3b,
e.g., in different wells. In some embodiments intact C3 and iC3b
are each detected using a polyclonal antibody. In some embodiments
the polyclonal antibody binds to C3. In some embodiments intact C3
is captured using an antibody that binds to C3a and is detected
using an antibody that binds to C3. In some embodiments intact C3
is captured using a monoclonal antibody that binds to C3a and is
detected using a polyclonal antibody that binds to C3. In some
embodiments intact C3 is captured using a monoclonal antibody that
binds to C3a and is detected using a polyclonal antibody that binds
to C3d. In some embodiments iC3b is captured using a monoclonal
antibody that binds to a neoepitope of iC3b and is detected using a
polyclonal antibody that binds to C3. In some embodiments total C3
is captured using a polyclonal antibody that binds to C3d and is
detected using a monoclonal antibody that binds to C3d. In some
embodiments intact C3 is captured using a monoclonal antibody that
binds to C3a and is detected using a polyclonal antibody that binds
to C3, and iC3b is captured using a monoclonal antibody that binds
to a neoepitope of iC3b and is detected using a polyclonal antibody
that binds to C3. In some embodiments the same polyclonal antibody
is used to detect intact C3 and to detect iC3b. In some embodiments
one or more capture antibody(ies) is/are attached to supports. In
some embodiments the sample comprises serum or plasma. In some
embodiments the sample comprises aqueous or vitreous humor. In some
embodiments the sample was obtained from a human subject. In some
embodiments the sample was obtained from a subject having or
suspected of having a complement deficiency or complement-mediated
disease or condition. In some embodiments the sample was obtained
from a subject suffering from AMD. In some embodiments the sample
was obtained from a subject who has recently suffered severe
injury. In some embodiments the sample was obtained from a subject
being treated with a complement inhibitor. In some embodiments the
sample comprises an agent being assessed for its ability to
modulate complement activation.
[0011] In another aspect the invention provides methods of
assessing complement activation in a subject comprising steps of:
(a) providing a biological sample obtained from the subject; and
(b) assessing complement activation in the sample according to an
inventive method described above or described elsewhere herein. In
some embodiments the method further comprises (c) using the result
of step (b) to provide diagnostic, prognostic, or treatment-related
information regarding the subject.
[0012] In another aspect, the invention provides a kit comprising:
(a) a capture agent that binds to intact C3 and does not bind to
C3b or iC3b; (b) a capture agent that binds to a neoepitope of
iC3b; and (c) a detection agent that binds to intact C3 and (d) a
detection agent that binds to iC3b. In some embodiments the capture
agent of (a) comprises a monoclonal antibody that binds to C3a. In
some embodiments the capture agent of (b) comprises a monoclonal
antibody that binds to a neoepitope of iC3b. In some embodiments
the detection agent of (c) and the detection agent of (d) are the
same. In some embodiments the detection agent that binds to intact
C3 comprises an antibody that binds to C3. In some embodiments the
antibody that binds to C3 is a polyclonal antibody. In some
embodiments the kit comprises (i) a polyclonal antibody that binds
to C3; and (ii) a monoclonal antibody that binds to C3a. In some
embodiments the kit comprises (i) a monoclonal antibody that binds
to a neoepitope of iC3b; and (ii) a monoclonal antibody that binds
to C3a. In some embodiments the kit comprises (i) a polyclonal
antibody that binds to C3; (ii) a monoclonal antibody that binds to
a neoepitope of iC3b; (iii) and a monoclonal antibody that binds to
C3a. The invention provides a kit comprising: (a) a capture agent
that binds to intact C3 and does not bind to C3b or iC3b; and (b) a
detection agent that binds to intact C3. In some embodiments the
capture agent of (a) comprises a monoclonal antibody that binds to
C3a. In some embodiments the monoclonal antibody is HM2075 or a
monoclonal antibody that binds to the same epitope as HM2075. In
some embodiments the monoclonal antibody is HM2073 or a monoclonal
antibody that binds to the same epitope as HM2073. In some
embodiments the detection agent of (b) comprises a polyclonal
antibody that binds to C3. In some embodiments the polyclonal
antibody does not bind to C3a. In some embodiments the polyclonal
antibody was raised against C3d. In some embodiments a kit of the
invention further comprises at least one item selected from the
group consisting of: instructions for use of the kit; one or more
native complement components or cleavage products; human serum
complement, optionally characterized for levels of one or more
complement components or cleavage products; serum depleted of one
or more complement components; a detectably labeled secondary
antibody; an enzyme substrate; a buffer; and a support. In some
embodiments the detectably labeled secondary antibody is labeled
with an enzyme. In some embodiments the kit comprises one or more
proteins selected from the group consisting of: C3 protein; C3d
polypeptide; C3b polypeptide; and iC3b polypeptide. Instructions
for use of the kit can comprise, e.g., instructions for assessing
the level of intact C3, instructions for assessing the level of
iC3b, instructions for assessing the level of total C3,
instructions for assessing complement activation. Instructions can
also or alternately comprise information regarding interpretation
of the results of performing an assay using the kit. For example,
such instructions may indicate that an increased level of intact C3
in a serum, plasma, or blood sample obtained from a subject is
indicative that the subject has or is likely to develop clinically
evident AMD.
[0013] Unless otherwise stated, the invention makes use of standard
methods of molecular biology, immunology, antibody production,
etc., and uses art-accepted abbreviations and meanings of terms.
This application refers to various patents and publications. The
contents of all scientific articles, books, patents, patent
applications, and other publications, mentioned in this application
are incorporated herein by reference. In addition, the following
publications are incorporated herein by reference: Current
Protocols in Molecular Biology, Current Protocols in Immunology,
Current Protocols in Protein Science, and Current Protocols in Cell
Biology, all John Wiley & Sons, N.Y., edition as of July 2002;
Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory
Manual, 3.sup.rd ed., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, 2001; Kuby Immunology, 6.sup.th ed., Kindt, T. J.,
et al (eds.), W. H. Freeman, 2006, Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 11.sup.th Ed., McGraw Hill,
2005, Katzung, B. (ed.) Basic and Clinical Pharmacology,
McGraw-Hill/Appleton & Lange; 10th edition, 2006; Goldman &
Ausiello, Cecil Textbook of Medicine, 23rd ed., W. B. Saunders,
2007; Paul, W. (ed.) Fundamental Immunology, 6.sup.th edition,
Lippincott Williams and Wilkins, Philadelphia, Pa. 2008.
BRIEF DESCRIPTION OF THE DRAWING
[0014] FIG. 1 is a schematic diagram showing steps in the pathway
of C3 activation and inactivation. FIG. 1a shows intact C3,
composed of beta and alpha chains linked by a disulfide bond. The
alpha chain has an intramolecular disulfide bond. C3 convertases
cleave intact C3 to yield C3a and C3b (FIG. 1b). Further cleavage
removes C3f, resulting in iC3b, which is inactive (FIG. 1c). Next,
C3dg is removed from iC3b, resulting in C3c (FIG. 1d). C3dg is
cleaved into C3d and C3g fragments (FIG. 1e).
[0015] FIG. 2a shows results of an ELISA assay in which anti-human
C3a mAb A203 (Quidel) was used as capture antibody. The ELISA plate
was coated with 100 ng/well mouse anti-C3a. FIG. 2b shows results
of an ELISA assay in which anti-human C3a mAb HM 2075 (Cell
Science) was used as capture antibody. The plate was coated with 50
ng/well mouse anti-C3a. FIG. 2c shows results of an ELISA assay in
which anti-human C3a mAb HM 2073 (Cell Science) was used as capture
antibody. The plate was coated with 100 ng/well mouse anti-C3a. In
each case, goat anti-C3-HRP was used as detection antibody.
[0016] FIG. 3a shows results of an ELISA assay in which anti-human
C3a mAb HM 2075 (Cell Science) was used as capture antibody. The
plate was coated with 100 ng/well mouse anti-C3a. FIG. 3b shows the
raw and processed data in Tables 2 and 3, respectively. FIG. 3c
shows results of an ELISA assay in which anti-human C3a mAb HM 2073
(Cell Science) was used as capture antibody. The plate was coated
with 100 ng/well mouse anti-C3a. FIG. 3d shows the raw and
processed data shown in Tables 4 and 5, respectively. In each case,
goat anti-C3-HRP was used as detection antibody.
[0017] FIGS. 4a and 4b show detection of intact C3 in the presence
of C3b and iC3b, showing specificity of the assay.
[0018] FIG. 5 shows a set of graphs demonstrating specificity of
the iC3b assay. Equal amounts of human purified complement proteins
C3, C3b and iC3b were used in the assay. The starting concentration
of each component is 10 .mu.g/ml in the leftmost well. Each sample
was then diluted twofold per well across the plate.
[0019] FIG. 6 shows results of the iC3b assay using mixed C3, C3b
and iC3b protein. Each sample was diluted twofold per well across
the plate.
[0020] FIG. 7 shows a comparison of iC3b standard curves in
blocking buffer. The concentration of iC3b was 2.5 .mu.g/ml in the
leftmost wells of the plate except for the 0.1% serum
condition.
[0021] FIG. 8a shows detection of iC3b in human vitreous samples to
which iC3b was added. FIG. 8b shows the raw and processed data in
Tables 6 and 7, respectively.
[0022] FIG. 9 is a graph showing levels of intact C3 in serum
samples from subjects with AMD and in serum samples from control
subjects more than 60 years of age.
[0023] FIGS. 10a and 10b are graphs showing levels of intact C3 in
serum samples from subjects with AMD and in serum samples from
control subjects more than 60 years of age. The samples included
the same samples used to generate the data shown in FIG. 9 and
several additional samples.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
[0024] This invention is not limited to particularly exemplified
systems or parameters as such may, of course, vary. The terminology
used herein is for the purpose of describing particular embodiments
of the invention and is not intended to limit the scope of the
invention in any manner. The definitions below are provided for the
convenience of the reader and are not intended to conflict with the
usage of such terms in the art unless specifically indicated.
I. DEFINITIONS
[0025] "Analyte" means any entity, particularly a chemical,
biochemical or biological entity to be assessed, e.g., whose amount
(e.g., concentration or mass), activity, composition, or other
property(ies) is/are to be detected, measured, quantified,
evaluated, analyzed, etc. An "analyte" can be a single molecular
species or can be composed of multiple distinct molecular
species.
[0026] "Antibody" encompasses intact and/or full length
immunoglobulins of types IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4),
IgE, IgD, IgM, IgY, antigen-binding fragments or single chains of
complete immunoglobulins (e.g., single chain antibodies, Fab
fragments, F(ab')2 fragments, Fd fragments, scFv (single-chain
variable), and dAb fragments), and other proteins that include at
least one antigen-binding immunoglobulin variable region, e.g., a
protein that comprises an immunoglobulin variable region, e.g., a
heavy (H) chain variable region (VH) and a light (L) chain variable
region (VL). The light chains of an antibody may be of type kappa
or lambda. An antibody may be polyclonal or monoclonal. A
polyclonal antibody contains immunoglobulin molecules that differ
in sequence of their complementarity determining regions (CDRs)
and, therefore, typically recognize different epitopes of an
antigen. Often a polyclonal antibody is derived from multiple
different B cell lines each producing an antibody with a different
specificity. A polyclonal antibody may be composed largely of
several subpopulations of antibodies, each of which is derived from
an individual B cell line. A monoclonal antibody is composed of
individual immunoglobulin molecules that comprise CDRs with the
same sequence, and, therefore, recognize the same epitope (i.e.,
the antibody is monospecific). Often a monoclonal antibody is
derived from a single B cell line or hybridoma. An antibody may be
a "humanized" antibody in which for example, a variable domain of
rodent origin is fused to a constant domain of human origin or in
which some or all of the complementarity-determining region amino
acids often along with one or more framework amino acids are
"grafted" from a rodent, e.g., murine, antibody to a human
antibody, thus retaining the specificity of the rodent
antibody.
[0027] "Approximately" or "about" in reference to a number
generally include numbers that fall within .+-.10%, in some
embodiments.+-.5%, in some embodiments.+-.1%, in some
embodiments.+-.0.5% of the number unless otherwise stated or
otherwise evident from the context (except where such number would
impermissibly exceed 100% of a possible value).
[0028] "Assay composition" refers to a composition on which an
inventive method for assessing complement activation is performed,
i.e., a composition whose concentration of intact C3 and total C3
is measured. An assay composition often comprises a biological
sample and one or more fluid diluents, such as water. A variety of
other substances may be included in an assay composition such as
salts, solvents, buffers, detergents, neutral proteins, e.g.
albumin, etc., which may be used to facilitate specific binding
interactions, maintain protein stability, reduce non-specific or
background interactions, etc. An assay composition may also
comprise one or more specific binding agents for use in carrying
out detection and/or one or more substances that modulate, e.g.,
activate or inhibit, one or more complement pathways.
[0029] "Biological sample" refers to a sample that contains or
consists of material obtained or derived from an organism,
typically a living organism that has a complement system, e.g., a
mammal, e.g., a human, non-human primate, rodent (e.g., mouse,
rabbit, rat), dog, cat, or other domesticated animal or bird, etc.
A biological sample can comprise, for example, any of the
following: whole blood, fractionated blood, plasma, serum, other
blood fraction or blood product, aqueous humor, vitreous humor,
tears, synovial fluid, cerebrospinal fluid, saliva, mucous, sweat,
milk, amniotic fluid, genital fluid, urine, fecal material, ascites
fluid, pericardial fluid, gastric fluid, peritoneal fluid, pleural
fluid, cyst fluid, broncheolar lavage fluid, nasal lavage,
lymphatic fluid, mammary fluid, duct fluid, bone marrow, tears,
prostatic fluid, tissue extract, glandular secretion, tissue
extract, whole cell lysate or fraction thereof, cell or tissue
extract, tissue homogenate, or cell culture supernatant. In some
embodiments a biological sample is essentially cell-free. In some
embodiments a biological sample contains no more than 1% cells by
weight or volume. The term "biological sample" can also refer to a
sample derived by processing a biological sample comprising any of
the afore-mentioned materials, e.g., by fractionating or diluting
the sample, isolating or purifying one or more substances (e.g.,
proteins) from the sample, etc.
[0030] "Detectable label" refers to a moiety that facilitates the
direct or indirect detection and/or quantitative or relative
measurement of a molecule to which it is attached. A detectable
label often has a property such as fluorescence, chemiluminescence,
radioactivity, color, magnetic or paramagnetic properties, etc.,
that renders it detectable, e.g., by the use of instruments that
detect fluorescence, chemiluminescence, radioactivity, color,
magnetic field, magnetic resonance, etc., or in some cases by
visual inspection. The label may be, e.g., fluorescent substance;
pigment; chemiluminescent or luminescent substance; colored
substance; magnetic substance; or a non-magnetic metal particle
such as gold colloid; a radioactive substance such as I-125, I-131,
P-32, H-3, S-25, or C-14. A fluorescent or chemiluminescent label
may be a fluorescein or a derivative thereof such as fluorescein
isothiocyanate; rhodamine or a rhodamine derivative such as
rhodamine isothiocyanate or tetramethyl rhodamine isothiocyanate; a
dancyl chloride (5-(dimethylamino)-1-naphthalenesulfonyl chloride);
a dancyl fluoride; a fluorescamine (4-phenylspiro[furan-2(3H);
1-(3H)-isobenzofuran]-3; 3-dione); a phycobiliprotein such as a
phycocyanine or phycoerythrin; an acridinium salt; a luminal,
lumiferin, luciferase, or aequorin; an imidazole; an oxalic acid
ester; a chelate compound of a rare earth element such as europium
(Eu), terbium (Tb) or samarium (Sm); or coumarin or a derivative
thereof such as 7-amino-4-methylcoumarin. Other examples include
derivatives of the phorphyrins, anthraquinones, anthrapyrazoles,
perylenequinones, xanthenes, cyanines, acridines, phenoxazines and
phenothiazines. In other embodiments the detectable label is a
quantum dot. In some embodiments a detectable label may comprise an
enzyme that acts on a substrate to produce a detectable substance.
The enzyme may be a dehydrogenase; an oxidoreductase such as a
reductase or oxidase; a transferase that catalyzes the transfer of
functional groups, such as an amino; carboxyl, methyl, acyl, or
phosphate group; a hydrolase that may hydrolyzes a bond such as
ester, glycoside, ether, or peptide bond; a lyases; an isomerase;
or a ligase. Examples include horseradish peroxidase, acid or
alkaline phosphatase, etc. The enzyme may also be conjugated to
another enzyme. The enzyme acts on a suitable substrate to produce
a detectable substance. For example, para-Nitrophenylphosphate
(pNPP) is a chromogenic substrate for acid and alkaline
phosphatase. When the detectable label is an alkaline phosphatase,
a suitable substrate may be an umbelliferone derivative, e.g.,
4-methyl-umbellipheryl phosphate. Tetramethyl benzidine (TMB) is a
substrate for horseradish peroxidase. In some embodiments the label
may be a hapten, such as adamantine, biotin, or carbazole. The
hapten may allow the formation of an aggregate when contacted with
a multi-valent antibody or (strep)avidin containing moiety. The
hapten may also allow easy attachment of a molecule to which it is
attached to a solid substrate.
[0031] "Detecting" or "detection" refer to determining the presence
of an analyte and often include providing a quantitative indication
of the concentration or amount of the analyte.
[0032] "Epitope" refers to the minimum portion of a molecule that
is recognized by, and thus determines the immunospecificity of, an
antibody that binds to such epitope. The term is also used herein
to refer to the minimum portion of a molecule that is recognized by
a non-antibody specific binding agent. Unless otherwise indicated,
it is assumed herein that a specific binding agent that binds to a
complement protein binds to an epitope present and accessible for
binding in the native protein, i.e., the epitope is not a
neoepitope. In some embodiments the epitope does not undergo
substantial structural change upon cleavage of a full length
protein, so that it retains the ability to be bound by a specific
binding agent that binds to the native protein. In some embodiments
the affinity of such binding is not substantially affected by
cleavage. For example, in some embodiments a specific binding agent
binds to a C3d epitope that does not undergo substantial structural
change upon cleavage of C3, C3b, iC3b, and/or C3dg.
[0033] "Isolated" means 1) separated from at least some of the
components with which it is usually associated in nature; 2)
prepared or purified by a process that involves the hand of man;
and/or 3) not occurring in nature.
[0034] Two or more moieties are "linked" if they are physically
associated or connected with one another to form a molecular
structure that is sufficiently stable so that they remain
associated under the conditions in which the linkage is formed and,
preferably, under the conditions in which the molecular structure
thus formed is used. In certain embodiments of the invention the
linkage is a covalent linkage. In other embodiments the linkage is
noncovalent. Moieties may be linked either directly or indirectly.
When two moieties are directly linked, they are either covalently
bonded to one another or are in sufficiently close proximity such
that intermolecular forces between the two moieties maintain their
association. When two moieties are indirectly linked, they are each
linked either covalently or noncovalently to a third moiety, which
maintains the association between the two moieties. In general,
when two moieties are referred to as being linked by a "linker" or
"linking moiety" or "linking portion", the linkage between the two
linked moieties is indirect, and typically each of the linked
moieties is covalently bonded to the linker. The linker can be any
suitable moiety that reacts with the two moieties to be linked
within a reasonable period of time, under conditions consistent
with stability of the moieties (which may be protected as
appropriate, depending upon the conditions), and in sufficient
amount, to produce a reasonable yield.
[0035] "Matrix" as used herein, refers to a substance that contains
complement component(s) or cleavage product(s) to be detected. A
matrix can comprise any of the substance(s) listed above in the
definition of "biological sample". A matrix can comprise any fluid
in which proteins can be dissolved or suspended. Often a matrix
comprises an aqueous medium.
[0036] "Neoepitope" is used herein as in the art to refer to an
epitope that is generated or becomes detectable as a result of
proteolytic cleavage of a complement component or cleavage
product.
[0037] "Plurality" means more than one.
[0038] "Polypeptide", as used herein, refers to a polymer of amino
acids, optionally including one or more amino acid analogs. A
protein is a molecule composed of one or more polypeptides. A
peptide is a relatively short polypeptide, typically between about
2 and 60 amino acids in length, e.g., between 8 and 40 amino acids
in length. The terms "protein", "polypeptide", and "peptide" may be
used interchangeably. Polypeptides used herein may contain amino
acids such as those that are naturally found in proteins, amino
acids that are not naturally found in proteins, and/or amino acid
analogs that are not amino acids. As used herein, an "analog" of an
amino acid may be a different amino acid that structurally
resembles the amino acid or a compound other than an amino acid
that structurally resembles the amino acid. A large number of
art-recognized analogs of the 20 amino acids commonly found in
proteins (the "standard" amino acids) are known. One or more of the
amino acids in a polypeptide may be modified, for example, by the
addition of a chemical entity such as a carbohydrate group, a
phosphate group, a farnesyl group, an isofarnesyl group, a fatty
acid group, a linker for conjugation, functionalization, or other
modification, etc. Certain non-limiting suitable analogs and
modifications are described in PCT publications WO2004026328 and
WO2007062249. The polypeptide may be acetylated, e.g., at the
N-terminus and/or amidated, e.g., at the C-terminus. The
modifications can occur anywhere in a polypeptide, including the
peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. A given polypeptide may contain many types of
modifications. Polypeptides may be branched and/or cyclic, with or
without branching. Polypeptides may, for example, be purified from
natural sources, produced in vitro or in vivo in suitable
expression systems using recombinant DNA technology in suitable
expression systems (e.g., by recombinant host cells or in
transgenic animals or plants), synthesized through chemical means
such as conventional solid phase peptide synthesis and/or methods
involving chemical ligation of synthesized peptides (see, e.g.,
Kent, S., J Pept Sci., 9(9):574-93, 2003), or any combination of
the foregoing.
[0039] "Reactive functional groups" as used herein refers to groups
such as, but not limited to, olefins, acetylenes, alcohols,
phenols, ethers, oxides, halides, aldehydes, ketones, carboxyl
groups, hydroxyl groups, carboxylic acids, esters, amides,
cyanates, isocyanates, thiocyanates, isothiocyanates, amines,
hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro,
nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones,
sulfonic acids, sulfinic acids, acetals, ketals, anhydrides,
sulfates, sulfenic acids isonitriles, amidines, imides, imidates,
nitrones, hydroxylamines, oximes, hydroxamic acids thiohydroxamic
acids, allenes, ortho esters, sulfites, enamines, ynamines, ureas,
pseudoureas, semicarbazides, carbodiimides, carbamates, imines,
azides, azo compounds, azoxy compounds, and nitroso compounds.
Reactive functional groups also include those frequently used to
prepare bioconjugates, e.g., N-hydroxysuccinimide esters,
maleimides, sulfhydryls, and the like (see, for example, Hermanson,
G., Bioconjugate Techniques, Academic press, San Diego, 1996).
Methods to prepare each of these functional groups are well known
in the art and their application to or modification for a
particular purpose is within the ability of one of skill in the art
(see, for example, Sandler and Karo, eds., "Organic Functional
Group Preparations", Academic Press, San Diego, 1989).
[0040] A first molecule is said to "specifically bind" to a second
molecule if it binds to the second molecule with substantially
greater affinity than to most or all other molecules present and/or
if the two molecules bind with an equilibrium dissociation
constant, K.sub.d, of 10-.sup.4 M or less, e.g., 10.sup.-5 M or
less, e.g., 10.sup.-6 M or less, 10.sup.-7M or less, 10.sup.-8 M or
less, or 10.sup.-9M or less under the conditions used. It will be
understood that if a first molecule binds specifically to a
particular epitope within a second molecule it may also bind
specifically to other molecules that contain the same epitope,
e.g., fragments of the second molecule that contain the epitope or
larger molecules that contain the second molecule.
[0041] "Specific binding agent" refers to a molecule or molecular
complex that specifically binds to a second molecule or molecular
complex. The second molecule or molecular complex is sometimes
referred to as a "target".
[0042] "Specific binding pair" refers to two molecules that
specifically bind to one another. Examples are biotin-avidin,
antibody-antigen, complementary nucleic acids, receptor-ligand,
etc.
[0043] "Subject", as used herein, refers to an individual from whom
a biological sample is obtained and/or on whom a test is performed
or to whom an agent is administered or on whom a test is performed,
e.g., for experimental, diagnostic, and/or therapeutic purposes.
Unless otherwise indicated, subjects are mammals, e.g., humans,
non-human primates, domesticated mammals such as dogs, or cats,
rodents, such as rabbits, mice, etc. Typically the subject is
human.
[0044] "Support" refers to any type of solid or semisolid physical
structure or group of structures to which a binding agent can be
attached. Typically a support has a rigid or semi-rigid surface or
surfaces. In some embodiments at least one surface of the support
is substantially planar while in other embodiments the surface is
curved or irregular. Exemplary supports can be plates, e.g.,
microtiter plates (sometimes called multiwell plates), having,
e.g., 96, 384, or 1536 wells, tubes (e.g., microfuge tubes),
dishes, slides, filters, fibers, wires, rods, pins, slides, etc. In
many embodiments, it may be desirable to analyze multiple aliquots
of a sample, or multiple samples, in physically separated regions
such as in individual wells.
[0045] A support can be made out of a wide variety of organic or
inorganic materials or mixtures thereof and can have a variety of
different shapes and sizes. Exemplary materials that may be used in
the manufacture of suitable supports are polymeric materials, e.g.,
plastics, such as polypropylene, polystyrene, poly(meth)acrylates,
polybutadienes, and the like, individually or in the form of
copolymers or blends. Exemplary inorganic support materials are
silicon oxide, silicon, mica, glass, quartz, titanium oxide,
vanadium oxide, metals such as gold or silver, alloys such as
steel, etc. A support can comprise a plurality of particles, often
referred to as beads, resins, microspheres, or other geometric
configurations. Particles may be microscopic or macroscopic and can
have a variety of different shapes, e.g., spheres, spheroids,
ellipsoids, etc. In some embodiments the particles average between
about 20 nm and about 20 microns in diameter (or longest axis or
other relevant dimension). In some embodiments the density of the
particles is about 0.5 mg/ml to about 2.0 mg/ml. Particles may be
made of a variety of materials, e.g., organic polymers (e.g.,
polystyrene), agarose, metals, etc., and may be homogeneous or
heterogeneous in composition. The particles may be non-magnetic or
magnetic. The term "magnetic" encompasses ferrimagnetic,
ferromagnetic, paramagnetic, and superparamagnetic materials.
Magnetic particles may comprise one or more materials selected from
the group consisting of: iron, cobalt, nickel, niobium, magnetic
iron oxides, iron hydroxides, and mixtures of any of the foregoing.
Often magnetic particles comprise smaller particles of a magnetic
material embedded in or encapsulated in a polymer material.
Magnetic particles may be conveniently recovered from an assay
composition by applying a suitable magnetic field, e.g., using a
magnet, as known in the art. The particle surface may be
functionalized with a moiety that comprises a reactive functional
group or specific binding pair member so as to facilitate binding
or attachment, either directly or indirectly, of a specific binding
agent to the particle.
II. ASSAYS FOR INTACT C3, iC3B, TOTAL C3, OR COMPLEMENT
ACTIVATION
[0046] The present invention provides compositions, methods, and
kits of use to assess the level of intact complement component C3
in a sample. The invention also provides compositions, methods, and
kits of use to assess the level of iC3b or total C3 in a sample. In
some aspects, the invention provides compositions, methods, and
kits of use to assess complement activation. The invention also
provides methods of diagnosing or monitoring subjects at risk of or
suffering from complement-mediated disorders or conditions or
complement deficiencies using an inventive assay described herein.
Certain of the methods comprise assessing the level of intact C3 in
a sample comprising serum, plasma, or blood obtained from the
subject. The invention further provides methods of selecting
appropriate therapy for a subject based at least in part on results
of an inventive complement activation assay or an inventive assay
for intact C3 described herein. Surprisingly, it was found that
serum levels of intact C3 can be used to distinguish individuals
with age-related macular degeneration (AMD), from individuals not
having AMD. Individuals who had been diagnosed AMD were found to
have significantly higher levels of intact C3 in their serum than
individuals not having AMD (see Example 11). Accordingly, in some
aspects, the invention provides methods of assessing a subject who
has or may develop age-related macular degeneration (AMD). The
invention also provides methods for deciding whether or when to
administer therapy to a subject suffering from or at risk of
developing AMD.
[0047] Certain aspects of the present invention are based at least
in part on measuring the level of intact complement component 3
(C3). Certain aspects of the present invention are based at least
in part on measuring activation of C3. C3 is the central protein of
the complement system. C3 is synthesized as a single polypeptide
chain. After removal of its signal sequence the pro-C3 polypeptide
is post-translationally modified by proteolytic cleavage into two
polypeptides (alpha and beta chains) linked by a disulfide bond to
yield mature C3. This form of C3 is referred to herein as "intact
C3" (FIG. 1a). The term "intact C3" is used interchangeably herein
with "full length C3", "native C3", "uncleaved C3", "unactivated
C3", or "unreacted C3". It will be appreciated that the term
"native C3" is sometimes reserved for intact C3 molecules that have
not undergone the first step of alternative pathway activation,
i.e., hydrolysis of the thioester bond (or in which the thioester
bond has reformed) and does not include the resulting
conformational intermediate or the form into which this
intermediate can be converted by an irreversible conformational
transition (Nishida, N., et al. Proc. Natl. Acad. Sci.
103(52):19737-42, 2006). However, in most embodiments of this
invention, these forms of mature C3 are detected together with
uncleaved C3, so the term "native C3" will be considered equivalent
to "uncleaved C3" unless otherwise indicated. During complement
activation intact C3 is cleaved by C3 convertase, releasing the C3a
fragment (77 amino acids in humans) and the much larger C3b
fragment (294 amino acids in humans) (FIG. 1b). See, e.g., Paul,
W., supra and Nishida N, et al, Proc. Natl. Acad. Sci.
103(52):19737-42, 2006. C3b is normally rapidly cleaved and
inactivated in vivo to the proteolytically inactive iC3b, releasing
the small fragment C3f (FIG. 1c). iC3b is subsequently cleaved to
form C3dg and C3c (FIG. 1d). Finally, C3dg is cleaved, giving rise
to C3d and the much smaller fragment C3g (FIG. 1e). For purposes of
the invention the terms "cleavage product", "split product", or the
like refer to the specific, named complement protein fragments
known in the art, a number of which are discussed herein (e.g.,
C3a, C3b, iC3b, C3dg, C3d, C3f, C3g, C4a, C4b, C5a, C5b).
[0048] The inventive approach, in certain embodiments, involves
detecting intact C3. In some embodiments, an inventive method
involves detecting intact C3 and detecting one or more C3 cleavage
products. In most embodiments, "detecting" comprises quantifying
the concentration or amount of the protein(s) detected, e.g.,
intact C3. Complement activation results in a reduced amount of,
e.g., intact C3. In some aspects, complement activation results in
increasing the level of C3 cleavage products. If little or no
complement activation has occurred, the amounts of C3b and iC3b are
low. Complement activation results in increased conversion of C3 to
C3b, which is subsequently cleaved to yield iC3b. In certain
embodiments of the invention, the extent of complement activation
is determined at least in part by measuring the amount of iC3b,
with a greater amount of iC3b indicating a greater extent of
complement activation. In certain embodiments of the invention, the
amount of intact C3 is also measured, and the ratio of iC3b to
intact C3 is used as an indicator of the extent to which complement
activation has occurred. If little or no complement activation has
occurred, the amount of iC3b, and the amount of iC3b relative to
intact C3, measured in a sample will be lower than if extensive
complement activation has occurred. Thus a higher ratio of iC3b to
intact C3 is indicative of greater complement activation than a
lower ratio. In certain embodiments of the invention, the amount of
total C3 is measured, and the ratio of iC3b to total C3 is used as
an indicator of the extent to which complement activation has
occurred, with a higher ratio of iC3b to total C3 being indicative
of greater complement activation than a lower ratio. It will be
appreciated that a ratio, e.g., a ratio of iC3b to intact C3, can
be expressed in a variety of ways. For example, a ratio can be
expressed as a number (which may be greater than, equal to, or less
than 1), fraction, or quotient (e.g., concentration of iC3b divided
by concentration of intact C3), or as otherwise known in the art.
It will also be appreciated that determining a ratio of two values
provides a determination of the reciprocal of the ratio. For
purposes of the present invention "determining the ratio of iC3b to
intact C3" encompasses determining any ratio or value that provides
the relative amounts or concentrations of iC3b and intact C3, and
"determining the ratio of iC3b to total C3" encompasses determining
any ratio or value that provides the relative amounts or
concentrations of iC3b and total C3.
[0049] It was found that certain complement component(s) and
cleavage product(s) can give different signals when measured in
different matrices and that various matrices themselves can
interfere with detection. In certain embodiments the inventive
methods produce accurate results when used to detect intact C3 or
iC3b in samples comprising different biologically relevant
matrices. In certain embodiments the inventive methods produce
accurate results when used to detect complement activation in
samples comprising different biologically relevant matrices. In
certain embodiments the inventive methods are highly specific for
detecting intact C3 versus C3b, iC3b, or C3d. In certain
embodiments the inventive methods show superior reproducibility and
the results exhibit less variation between individual users and
between tests performed on the same sample on different days than
is the case for a variety of commercially available and published
assays for complement activation and/or for a complement component
or cleavage product. In certain embodiments the inventive methods
provide more consistent results when complement activation and/or a
complement component or cleavage product is measured in different
matrices, e.g., phosphate buffered saline (PBS), serum, vitreous,
or blocking buffer, than is the case for a variety of commercially
available and published assays for complement activation.
[0050] According to certain embodiments of the invention, the
magnitude of the difference between the amounts of total C3 and
intact C3 provides an indication of the extent to which complement
activation has occurred. "Total C3" encompasses C3 present
collectively in the five states shown in FIGS. 1b-1e. In some
embodiments, detecting total C3 comprises detecting intact C3 and
those cleavage products of C3 that comprise C3d. In these
embodiments detecting total C3 comprises full length C3, C3b, iC3b
C3dg, and C3d but does not comprise detecting C3a, C3c, C3f, and
C3g as individual fragments. Detecting full length C3, C3b, and
iC3b accounts for C3 molecules in states shown in FIGS. 1a-1c,
while detecting C3dg and C3d accounts for C3 molecules in states 1d
and 1e, respectively. In some embodiments, detecting total C3
comprises intact C3 and detecting those cleavage products of C3
that comprise C3c. In these embodiments detecting total C3
comprises full length C3, C3b, iC3b, and C3c but does not comprise
C3a, C3d, C3dg, C3f, or C3g as individual fragments. Detecting full
length C3, C3b, and iC3b accounts for C3 molecules in states shown
in FIGS. 1a-1c, while detecting C3c accounts for C3 molecules in
states 1d and 1e. If little or no complement activation has
occurred, the amounts of intact C3 and total C3 in a sample are
substantially identical, while if extensive complement activation
has taken place, the amount of intact C3 will be much lower than
that of total C3. For example, consider a sample that contains 100
unactivated C3 molecules and sufficient amounts of the upstream
complement components. At time t=0 a complement activating agent is
added. Over time, complement activation occurs and molecules of
intact C3 are cleaved and pass through states shown in FIGS. 1b-1e.
For example, at t=5, there might be 50 intact C3 molecules, 25
molecules in state 1b, 20 in state 1c, and 5 in state 1d, at which
point it may be said that 50% complement activation has occurred.
At t=10, there might be 25 intact C3 molecules, 25 molecules in
state 1b, 20 molecules in state 1c, and 15 molecules in each of
states 1d and 1f, at which point it may be said that 75% complement
activation has occurred. Eventually all C3 molecules will have been
cleaved, at which point it may be said that 100% complement
activation has occurred. Of course in a living subject, ongoing
synthesis of C3 may occur, and the results of an inventive assay
may reflect the consequences of both synthesis and consumption.
[0051] Amounts (i.e., levels) of intact C3, iC3b, and/or total C3
are typically expressed in terms of concentration but may be
expressed in terms of mass or weight. Concentration may be
expressed in various ways, e.g., in terms of molarity, molality,
mole fraction, mass fraction (mass of a substance in a mixture as a
fraction of the mass of the entire mixture), mass per unit volume,
etc. For purposes of description herein, concentration (e.g., mass
per unit volume) will generally be used. One of skill in the art
can readily convert between concentration and moles or mass in a
given volume. The difference between the concentration of total and
intact C3 may be expressed in a variety of ways. In some
embodiments of the invention the difference is expressed in terms
of relative concentrations. For example, the difference between the
concentration of total C3 and intact C3 may be expressed as the
fraction of C3 that is intact, e.g., concentration of intact C3
divided by concentration of total C3 (which may be multiplied by
100 to obtain a percentage), or as the ratio of the concentration
of total C3 to the concentration of intact C3 or vice versa. In
some embodiments, the difference is expressed as an absolute
number.
[0052] It will be appreciated that an initial sample (e.g., a
sample obtained from a subject) may be divided into multiple
smaller samples, which are also considered "samples" but may at
times be referred to as sub-samples, or aliquots. In some
embodiments, a measurement is performed on multiple aliquots and
the results are averaged. For example, in some embodiments, intact
C3 is measured in more than one aliquot, and the results are
averaged to determine an intact C3 value for the sample. In some
embodiments, iC3b is measured in more than one aliquot and the
results are averaged to determine an iC3b value for the sample. The
ratio of the averages is then obtained in those embodiments of the
invention that involve determining the ratio of iC3b to intact C3.
In some embodiments, total C3 is measured in more than one aliquot,
and the results are averaged.
[0053] Biological samples may be diluted so that the analytes are
present in a suitable concentration to be accurately quantified. In
some embodiments a sample is diluted with an aqueous medium. In
some embodiments a sample is diluted with a blocking buffer. In
some embodiments the blocking buffer is a standard blocking buffer
useful in an ELISA assay to reduce non-specific binding of
detection antibodies. In some embodiments the blocking buffer is a
protein-free blocking buffer, e.g., cat. no. 37570, 37571 37572, or
37573 from Thermo Fisher Scientific, Inc. In some embodiments the
blocking buffer comprises one or more proteins. In most embodiments
the blocking buffer is free of C3 and C3 cleavage fragments. In
some embodiments a sample is diluted with phosphate buffered saline
or tris buffered saline. In some embodiments, a blood, plasma, or
serum sample is diluted so that it contains no more than 0.5%, no
more than 0.25%, no more than 0.1%, or no more than 0.05% blood,
plasma, or serum, respectively. In some embodiments, a sample
contains up to 0.5%, up to 0.25%, up to 0.1%, or up to 0.05% blood,
plasma, or serum. In some embodiments, a vitreous or aqueous humor
sample is diluted so that it contains no more than 40%, no more
than 30%, no more than 20%, or no more than 10% vitreous or aqueous
humor. In some embodiments, a sample contains up to 40%, up to 30%,
up to 20%, or up to 10% vitreous or aqueous humor.
[0054] In some embodiments, to obtain a sample, blood may be drawn
directly into tubes containing EDTA at a final concentration of at
least about 10 mM. In some embodiments, one or more protease
inhibitors are added to the samples or to the assay composition. In
some embodiments a complement inhibitor is added to the sample to
inhibit complement activation. In some embodiments a sample is
processed to remove one or more abundant proteins prior to
assessing levels of intact C3. For example, it may be desirable to
remove one or more abundant proteins selected from the group
consisting of: albumin, fibrinogen, transferrin, IgA, IgM, IgG,
.alpha.2-macroglobulin, .alpha.1-antitrypsin, haptoglobin, apo A-I,
apo A-II, LDL (ApoB), .alpha.1-acid glycoprotein, vitronectin,
.alpha.-crystallin and/or .beta.-crystallin from a blood, plasma,
serum, vitreous humor, aqueous humor, or CSF sample. Such depletion
can be accomplished, e.g., using immunoaffinity approaches in which
a sample is contacted with antibodies to the protein(s) whose
removal is desired. For example, Seppro.RTM. columns, microbeads,
and tips (available from Sigma-Aldrich) comprise chicken IgY
antibodies immobilized on a support and provide a convenient means
to remove one or more such proteins from a sample.
[0055] A variety of methods may be used to detect intact C3, iC3b,
and/or total C3. In many embodiments, the methods for detecting
intact C3 comprise (a) providing a sample; (b) providing a specific
binding agent that binds to intact C3; and (c) detecting binding of
the specific binding agent to analyte(s) present in the sample. In
many embodiments, the methods for detecting intact iC3b comprise
(a) providing a sample; (b) providing a specific binding agent that
binds to iC3b and substantially does not bind to intact C3 or to
C3b; and (c) detecting binding of the specific binding agent to
analyte(s) present in the sample. In many embodiments, binding is
detected in a quantitative manner, thereby providing an indication
of the amount of intact C3 or iC3b, respectively, present in the
sample. In some embodiments, the methods for detecting total C3
comprise (a) providing a sample; (b) providing a specific binding
agent that binds to full length C3 and to cleavage products that
contain C3d; and (c) detecting binding of the specific binding
agent to analyte(s) present in the sample. In some embodiments,
binding is detected in a quantitative manner, thereby providing an
indication of the amount of intact C3, iC3b, or total C3 present in
the sample. The phrase "specific binding agent that binds to intact
C3" is intended to indicate that the specific binding agent binds
to intact C3 and may bind to C3a but does not bind to other C3
cleavage products. For example, an antibody that binds to an
epitope within C3a (such epitope not being a neo-epitope) is
considered a specific binding agent that binds to intact C3, since
intact C3 comprises C3a. Such an agent would not, however, bind to
C3c, C3b, C3dg, or cleavage products thereof. A binding agent that
specifically binds to a particular complement component will in
many cases also bind to one or more cleavage products of the
component. For example, a monoclonal antibody that binds to an
epitope of C3 may bind most or all C3 cleavage products that
contain the epitope. A binding agent that specifically binds to a
cleavage product of interest will in many cases also bind to
products obtained by further cleavage of the cleavage product of
interest as well as to the full length complement component that
comprises the cleavage product of interest and any larger cleavage
products that comprise the cleavage product of interest. A specific
binding agent that binds to iC3b but substantially does not bind to
intact C3 or C3b may bind to at least some cleavage products of
iC3b, e.g., C3d. In certain embodiments of the invention a specific
binding agent that binds to a first analyte is said to "not
substantially bind" to a second analyte if one or more of the
following conditions is/are met: (i) a sample that contains equal
concentrations X of the first analyte and the second analyte
produces no more than 1.25 times as large a signal, or in some
embodiments no more than 1.1 times as large a signal, or in some
embodiments no more than 1.05 times as large a signal, as does a
sample that contains only the first analyte at concentration X when
the specific binding agent is used as a capture agent to quantitate
analyte(s) in the sample (assuming that the detection agent used is
able to detect the first and second analytes with approximately
equal efficiency or results are adjusted to account for varying
detection efficiency); (ii) a sample that contains equal
concentrations X of the first analyte and the second analyte
produces no more than 1.25 times as large a signal, or in some
embodiments no more than 1.1 times as large a signal, or in some
embodiments no more than 1.05 times as large a signal, as does a
sample that contains only the first analyte at concentration X when
the specific binding agent is used as a detection agent to
quantitate analyte(s) in the sample (assuming that the capture
agent used is able to capture the first and second analytes with
approximately equal efficiency or results are adjusted to account
for varying capture efficiency); (iii) when the specific binding
agent is used as a capture agent, a sample that contains a
concentration X of the second analyte produces no more than 25% of
the signal, or in some embodiments no more than 10% of the signal,
or in some embodiments no more than 5% of the signal produced by a
sample that contains the same concentration X of the first analyte
(assuming that the detection agent used is able to detect the first
and second analytes with approximately equal efficiency or results
are adjusted to account for varying detection efficiency); (iv)
when the specific binding agent is used as a detection agent, a
sample that contains a concentration X of the second analyte
produces no more than 25% of the signal, or in some embodiments no
more than 10% of the signal, or in some embodiments no more than 5%
of the signal produced by a sample that contains the same
concentration X of the first analyte (assuming that the capture
agent used is able to capture the first and second analytes with
approximately equal efficiency or results are adjusted to account
for varying capture efficiency). Specificity may be assessed over
or an analyte concentration range of interest or a portion thereof
(see Examples) and may be assessed using pure complement components
or cleavage products, e.g., in a matrix similar to that in which
binding would occur when the specific binding agent is used in an
assay of the invention. Background signal from a sample containing
the matrix alone (i.e., without added first or second analyte) may
be subtracted.
[0056] Native complement proteins or cleavage products may be
substrates for nonspecific proteases and/or other proteolytic or
other breakdown pathways in addition to, or instead of, those that
generate the cleavage products discussed herein. The term
"degradation products" refers to complement protein fragments,
e.g., C3 fragments, other than the characteristic cleavage products
specifically mentioned herein. In some embodiments of the invention
intact C3, iC3b or total C3 comprises at least some degradation
products. Whether or not a particular degradation product is
included within intact C3, iC3b, or total C3 in a given embodiment
of the invention will often depend at least in part on the
properties of the binding agent(s) used in that embodiment and is
not expected to significantly affect the results of the assay.
[0057] A variety of specific binding agents are of use in various
embodiments of the present invention. In many embodiments of the
invention the specific binding agent(s) are antibodies. Antibodies
can be monoclonal or polyclonal and can be prepared by techniques
that are well known in the art. Briefly, such methods include (i)
immunizing a host such as a rodent (mouse, rabbit, etc.), chicken,
goat, sheep, cow, or other animal with an antigen of interest and
collecting sera (for polyclonal antibodies); or (ii) preparing and
culturing (in vivo or in vitro) an immortalized hybrid cell line
wherein one of the cells produces an antibody of interest (obtained
from a host such as a mouse that has been immunized with or
otherwise exposed to an antigen of interest) and the other cell is
a myeloma cell and collecting the secreted protein (for monoclonal
antibodies). It will be appreciated that antibodies may be
collected from chicken eggs, milk, transgenic animals or plants
engineered to express an immunoglobulin, etc. In some embodiments a
host is immunized with a nucleic acid construct or virus that
encodes the antigen of interest. Antibodies (usually monoclonal)
can also be generated by cloning and expressing nucleotide
sequences or mutagenized versions thereof that encode at least the
amino acid sequences of an antibody required for specific binding
or by chemical synthesis (see, e.g., Allen, T., Nature Reviews
Cancer, Vol. 2, 750-765, 2002, and references therein). Such amino
acid sequences may be obtained from an antibody originally produced
by a host or may be generated and/or selected in vitro using
techniques such as phage display (Winter, G. et al., Annu. Rev.
Immunol. 12:433-455, 1994), ribosome display (Hanes, J., and
Pluckthun, A. Proc. Natl. Acad. Sci. USA. 94:4937-4942, 1997), etc.
To prepare antibodies for use in the present invention, complement
components (e.g., full length C3) or cleavage products (e.g., C3a,
C3d, etc.) may be used as antigens. Such proteins may be purified
from serum or produced recombinantly or by chemical synthesis. In
some embodiments a full length protein is used as an antigen while
in other embodiments a portion is used, e.g., a portion generated
by cleaving a full length component in vitro or in vivo. Peptides
(usually at least 8-10 amino acids in length, e.g., between 8 and
50 amino acids, and usually produced by chemical synthesis) whose
sequence is derived from that of a larger polypeptide may be used
and can be particularly useful if it is desired to generate an
antibody that binds to a particular region of a polypeptide. The
antigen may be conjugated to a carrier or otherwise modified prior
to immunizing the host. Antibodies may be purified from sera,
ascites fluid, or cell culture supernatant, etc., using well known
methods such as affinity purification, e.g., protein A/G affinity
purification and/or affinity purification using the antigen as an
affinity reagent. See, e.g., Kaser, M. and Howard, G., "Making and
Using Antibodies: A Practical Handbook" and Sidhu, S., "Phage
Display in Biotechnology and Drug Discovery", CRC Press, Taylor and
Francis Group, 2005.
[0058] A variety of non-immunoglobulin specific binding agents
could be used in various embodiments of the invention. For example,
anticalins are binding proteins that are constructed on the basis
of lipocalins as a scaffold (Skerra, J., J. Biotechnol.,
74(4):257-75, 2001). Affibodies are binding proteins generated from
combinatorial libraries constructed using the protein A-derived Z
domain as a scaffold (see, e.g., Nord K, Eur J Biochem.,
268(15):4269-77, 2001). Nucleic acid ligands, sometimes referred to
as aptamers, are oligonucleotides that bind specifically and with
high affinity to a target. They can comprise DNA, RNA, and/or
non-standard nucleotides, e.g., 2'-fluoro or 2'-O-methyl modified
nucleotides, and may be identified using, e.g., systematic
evolution of ligands by exponential enrichment (SELEX) or various
directed evolution techniques that are known in the art. See, e.g.,
Tuerk, C. and Gold, L., Science 249(4968): 505-10, 1990; Brody E N,
Gold L. J. Biotechnol., 74(1):5-13, 2000. Aptamers that bind to C3
have been identified. See, e.g., U.S. Pat. Pub. No. 20030191084.
The invention provides embodiments in which each type of specific
binding agent described herein is employed.
[0059] In some embodiments of the invention an ELISA assay is used
to detect intact C3, iC3b, and/or total C3. As used herein, the
term "ELISA assay" refers to any of a number of techniques that may
be used to detect the presence of an analyte of interest in a
sample. In ELISA assays the analyte of interest is directly or
indirectly immobilized on a support and is contacted with a first
specific binding agent that binds to the analyte and a detection
enzyme is used to detect the first specific binding agent. Suitable
detection enzymes are known in the art, and a number are mentioned
above. The detection enzyme is contacted with a substrate on which
it acts to generate a detectable signal. For example, the substrate
may be a chromogenic or fluorogenic substrate that generates a
colored or fluorescent moiety, when acted on by the enzyme, e.g.,
when cleaved by the enzyme. The detection enzyme may be linked to
the first specific binding agent. In many embodiments, however, the
detection enzyme is linked, e.g., covalently linked, to a second
specific binding agent that specifically binds to the first
specific binding agent. In some embodiments, the detection enzyme
is linked, e.g., covalently linked, to a third specific binding
agent that binds to the second specific binding agent. For example,
the second specific binding agent may be linked to biotin and the
detection enzyme linked to avidin or streptavidin.
[0060] A number of different ELISA assay types may be used. These
general assay formats are well known in the art. In a sandwich
ELISA, a first specific binding agent that binds to the analyte is
immobilized on a support and used to "capture" analyte present in
the sample. The first specific binding agent may be referred to as
a "capture agent". Analyte present in the sample binds to the first
specific binding agent and is thereby immobilized. Unbound material
may be removed. One or more washing steps may be performed. A
composition comprising a second specific binding agent specific for
the analyte is then contacted with the support. The second specific
binding agent serves as a "primary detection agent" and binds to
analyte that had been immobilized via binding to the first specific
binding agent. Unbound second specific binding agent is typically
removed. The second specific binding agent is then detected. In
some embodiments the second specific binding agent has an enzyme
linked thereto. In some embodiments the second specific binding
agent does not have an enzyme linked thereto but instead is
detected using a third specific binding agent ("secondary detection
agent") that binds to the second specific binding agent and has an
enzyme linked thereto. In some embodiments the primary detection
agent comprises an antibody and the secondary detection agent
comprises an antibody that binds to the antibody of the primary
detection agent (e.g., to the Fc domain). It will be appreciated
that in such cases it may be desirable to avoid using capture agent
and primary detection agent that comprise antibodies raised in the
same species in order to avoid the secondary detection agent
binding to the capture agent. In some embodiments the primary
detection agent comprises an antibody linked to a first member of a
specific binding pair, and the secondary detection agent comprises
an antibody linked to the other member of the specific binding
pair, thus enabling use of a secondary detection agent that does
not bind to the antibody portion of the primary detection agent.
For example, the primary detection agent may comprise an antibody
to which biotin has been conjugated, and the secondary detection
agent may comprise avidin or streptavidin conjugated to an enzyme
or may comprise a preformed complex between avidin or streptavidin
and a biotinylated enzyme. In another embodiment the primary
detection agent comprises an antibody conjugated to biotin, and the
secondary detection agent comprises avidin or streptavidin
conjugated to an enzyme or comprises a complex between avidin or
streptavidin and a biotinylated enzyme. Biotin/avidin and
biotin/streptavidin are exemplary specific binding pairs and that
others could be used in these embodiments.
[0061] Often the capture agent and the primary detection agent are
different. For example, it may be desirable to select capture and
primary detection agents that bind to different portions of an
analyte to avoid binding by the capture agent from interfering with
binding by the primary detection agent. In some embodiments, the
capture agent is a monoclonal antibody that binds to an analyte
(e.g., intact C3 or iC3b) and the primary detection agent is a
polyclonal antibody that binds to the analyte. In some embodiments,
the capture agent may be a polyclonal antibody that binds to an
analyte and the primary detection agent may be a monoclonal
antibody that binds to the analyte. The polyclonal antibody will
typically contain a plurality of antibody molecules that bind to
different epitopes of an antigen. Thus capturing an analyte using a
monoclonal antibody is not expected to interfere with detection
using a polyclonal antibody, and capturing an analyte using a
polyclonal antibody is not expected to interfere with detection
using a monoclonal antibody as a primary detection agent. In some
embodiments the capture agent and the primary detection agent are
monoclonal antibodies that bind to different epitopes of an
analyte.
[0062] In some embodiments of particular interest a monoclonal
antibody that specifically binds to the C3a portion of C3 and
substantially does not bind to C3 fragments that do not contain C3a
is used as a capture agent for intact C3. In some embodiments a
monoclonal antibody that specifically binds to a neoepitope of iC3b
and substantially does not bind to intact C3 or to C3b is used as a
capture agent for iC3b. In these embodiments of the invention a
polyclonal antibody that binds to C3 is often used as a detection
agent. The polyclonal antibody must contain at least some antibody
molecules that bind to iC3b. In some embodiments the anti-C3
polyclonal antibody is a goat antibody. In some embodiments the
anti-C3 polyclonal antibody is a chicken antibody. In some
embodiments horseradish peroxidase is conjugated to the
antibody.
[0063] An aspect of certain embodiments of the invention is the
selection of antibodies for use as capture and detection agents.
The inventors discovered that many published and commercially
available antibodies exhibited significant and unexpected crosstalk
between intact C3 and various C3 cleavage products or between
different C3 cleavage products. For example, certain monoclonal
antibodies against human C3a show significant and unexpected
cross-reactivity with C3b and iC3b. It was recognized that
cross-reactivity could be a significant source of inaccuracy in
certain situations. Of particular concern in developing an assay
for iC3b was crosstalk between intact C3 and iC3b observed with
many of the iC3b antibodies tested. Further testing showed that
crosstalk between C3b and iC3b was even more significant with at
least some of these antibodies. This was of concern because iC3b
levels are expected to be present at much lower levels than intact
C3 in patient samples. One aspect of certain embodiments of the
invention is the selection of antibodies with specificity for
intact C3 or iC3b so as to minimize such crosstalk.
[0064] In some embodiments of the invention, an antibody used to
capture or detect intact C3 is characterized in, when used as a
capture agent on samples that contain either intact C3, C3b, iC3b,
C3c, or C3d at a concentration of about 0.67 .mu.g/ml, followed by
detection using an anti-C3 antibody (e.g., goat anti-C3-HRP
antibody such as MP Biomedicals, cat. no. 55237), the signal from
the sample containing intact C3 is at least 9-fold as great as the
signal from samples containing either C3b, iC3b, C3c, or C3d. In
some embodiments of the invention, an antibody used to capture or
detect intact C3 is characterized in, when used as a capture agent
on samples that contain either intact C3, C3b, iC3b, C3c, or C3d at
a concentration of about 0.2 .mu.g/ml, followed by detection using
an anti-C3 antibody (e.g., goat anti-C3-HRP antibody such as MP
Biomedicals, cat. no. 55237), the signal from the sample containing
intact C3 is at least 15-fold as great as the signal from samples
containing either C3b, iC3b, C3c, or C3d. In some embodiments, an
antibody used to capture or detect intact C3 has approximately
equal or greater ability to distinguish intact C3 from C3b, iC3b,
C3c, and C3d in samples containing such proteins (e.g., at
concentrations and under conditions described in the relevant
Examples or Figures) as does anti-C3a monoclonal antibody clone 474
(cat. no. HM2073 Hycult Biotechnology, available from Cell
Sciences, Inc., Canton, Mass.) or clone 2898 (cat. no. HM 2075,
Hycult Biotechnology, available from Cell Sciences, Inc., Canton,
Mass.) when used as a capture agent or detection agent,
respectively. In some embodiments of the invention an antibody used
to capture or detect intact C3 binds to the same epitope of C3a as
does the mAb of clone 474 or clone 2898. A second mAb that binds to
the same epitope as a first mAb may be identified, e.g., by
screening a panel of mAbs raised against C3a to identify those that
compete with the first mAb for binding to C3a. One can clone the
genes encoding the heavy and light chains, or at least the
complementarity determining regions (CDRs) or variable domains of
the heavy and light chains from an antibody-producing cell clone.
The heavy and light chain genes can then be expressed in a
heterologous cell. The CDRs or variable domains, and optionally
additional amino acids, can be "grafted" to a different antibody,
e.g., can be used in place of the corresponding CDRs or variable
domains of such antibody, resulting in an antibody with equivalent
binding specificity.
[0065] In some embodiments of the invention, an antibody used to
capture or detect iC3b is characterized in that, when used as a
capture agent on samples that contain either iC3b, C3, or C3b at a
concentration of 10 .mu.g/ml, followed by detection using an
anti-C3 antibody, the signal from the sample containing iC3b is at
least 5-fold as great as that from the samples containing C3 or
C3b. In some embodiments of the invention, an antibody used to
capture or detect iC3b is characterized in that, when used as a
capture agent on samples that contain either iC3b, C3, or C3b at a
concentration of 5 .mu.g/ml, followed by detection using an anti-C3
antibody, the signal from the sample containing iC3b is at least
7.5-fold as great as that from the samples containing C3 or C3b. In
some embodiments of the invention, an antibody used to capture or
detect iC3b is characterized in that, when used as a capture agent
on samples that contain either iC3b, C3, or C3b at a concentration
of 2.5 .mu.g/ml, followed by detection using an anti-C3 antibody,
the signal from the sample containing iC3b is at least 15-fold as
great as that from the samples containing C3 or C3b. In some
embodiments, an antibody used to capture or detect iC3b has
approximately equal or greater ability to distinguish iC3b from
intact C3 and from C3b in samples containing such proteins (e.g.,
at concentrations and under conditions described in the relevant
Examples or Figures) as does anti-iC3b antibody catalog A209
(Quidel Corp., San Diego, Calif.) (Tamerius, J. D., M. Pangburn, et
al., Detection of a neoantigen on human iC3b and C3d by monoclonal
Antibody, J. Immunol. 135:2015, 1985) when used as a capture agent
or detection agent, respectively. In some embodiments of the
invention the antibody used to capture or detect iC3b binds to the
same epitope of iC3b as does mAb A209.
[0066] In some embodiments of the invention a polyclonal antibody
that specifically binds to C3d (as well as to intact C3 and all
cleavage products that contain C3d) is used as a capture or
detection agent for intact C3, iC3b, and/or total C3. In some
embodiments of the invention a polyclonal antibody that
specifically binds to C3 (and to all cleavage products that contain
C3d or C3c) is used as a capture agent or detection agent for
intact C3, iC3b, and/or total C3.
[0067] A variety of different peptides or C3 fragments could be
used as antigens for purposes of producing a polyclonal or
monoclonal antibody having desired binding properties, e.g., (i)
binds to C3d as well as to intact C3 and all cleavage products that
contain C3d; (ii) binds to intact C3 and all cleavage products that
contain C3c; (iii) binds to intact C3 but not to C3 cleavage
products that do not contain C3a; (iv) binds to iC3b but
substantially does not bind to intact C3 or C3b. In some
embodiments an antibody that binds to C3d is raised against an
antigen that comprises or consists of C3d. In some embodiments an
antibody that binds to C3d is raised against an antigen that
comprises or consists of at least half of C3d. In some embodiments
an antibody that binds to C3 is raised against an antigen that
comprises or consists of full length C3. In some embodiments an
antibody that binds to C3 is raised against an antigen that
comprises or consists of a portion of C3 that comprises at least
half of C3. In some embodiments the antibody that binds to intact
C3 but not to C3 cleavage products that do not contain C3a is
raised against an antigen that comprises or consists of C3a. In
some embodiments an antibody that binds to iC3b but substantially
does not bind to C3 or C3b is raised using iC3b or a portion
thereof (e.g., a portion that contains a new terminus generated by
cleavage of C3b) as an antigen. Antibodies can be screened to
identify those with desired binding properties.
[0068] Exemplary antibodies of use in certain embodiments of the
present invention are described in the Examples. In some
embodiments, an antibody that binds to the same epitope as an
antibody described in the Examples is used. One of skill in the art
can screen panels of monoclonal antibodies to identify those that
bind to the same epitope as another antibody. In some embodiments,
an antibody that has similar binding affinity for its target as an
antibody described in the Examples is used. Additional antibodies
that specifically bind to complement components such as C3a, C3d,
C3c, C3, etc., are commercially available.
[0069] In an indirect ELISA, the analyte is attached directly to
the support, e.g., by adsorption. Often other substances present in
the sample (e.g., other proteins) are also adsorbed. A first
specific binding agent that serves as a primary detection agent is
contacted with the support and binds to the analyte. Unbound
specific binding agent is removed and the support is washed. In
some embodiments the primary detection agent comprises a detection
enzyme, which is contacted with a substrate to detect the analyte.
Alternately a second specific binding agent that specifically binds
to the first specific binding agent is contacted with the support
and binds to the first specific binding agent. Unbound second
specific binding agent is removed. The second specific binding
agent has a detection enzyme linked thereto, which is used to
detect the analyte. In other embodiments, the second specific
binding agent does not comprise a detection enzyme. Instead, after
allowing the second specific binding agent to bind to the primary
detection agent, unbound second specific binding agent is removed
and the support is then contacted with a third specific binding
agent that binds to the second specific binding agent and has a
detection enzyme linked thereto, which is used to detect the
analyte.
[0070] In a competitive ELISA, a first specific binding agent that
recognizes the analyte is contacted with a sample containing
analyte. The resulting assay composition is maintained to allow
formation of binding agent/analyte complexes. The assay composition
is then contacted with a support that has analyte attached thereto.
Specific binding agent that is not in a complex with the analyte in
the assay composition binds to analyte attached to the support.
Unbound specific binding agent is then removed. Binding agent that
remains bound to the support is then detected using a second
binding agent to which an enzyme is bound. A substrate is added,
and the enzyme acts on the substrate to generate a detectable
signal. In this type of ELISA assay, the higher the analyte
concentration, the weaker the resulting signal.
[0071] While ELISA assays are of particular interest herein, other
methods of detecting intact C3, iC3b, and/or total C3 may be used
in certain embodiments of the invention. Many of these assays are
similar to ELISA assays in that they involve detecting an analyte
that has been immobilized on a support but use a detectable label
that is not an enzyme. For example, a specific binding agent used
for detection purposes may be labeled with a detectable label other
than an enzyme, e.g., a fluorescent or chemiluminescent substance
such as fluorescein, rhodamine, phycoerythrin, a quantum dot, etc.
For purposes of description the term "ELISA assay" should be
understood to encompass embodiments in which such non-enzyme
detectable labels are used.
[0072] ELISA assays may be performed in a variety of ways depending
at least in part on the nature of the support. For example, an
analyte or capture agent may be immobilized on the surface of a
receptacle such as a well of a multiwell plate, e.g., by adsorption
or via a reactive functional group. Reagents are added to the well
in a sequence of steps separated by washing steps in which unbound
reagents are removed. Alternately, reagents can be placed in
different receptacles and an analyte or capture agent immobilized
on a surface that can be transferred from one receptacle to
another, with washing steps between transfers. For example, the
analyte could be immobilized on a rod or pin, which is dipped
sequentially into a receptacle containing a first specific binding
agent, a receptacle containing a second specific binding agent that
binds to the first specific binding agent, and then a receptacle
containing an enzyme substrate (with wash steps in between). In
another embodiment the capture agent is immobilized on a rod or pin
which is dipped into a receptacle containing a sample and then
sequentially into a receptacle containing a second specific binding
agent, and a receptacle containing an enzyme substrate (with wash
steps in between).
[0073] In some embodiment a particle-based ELISA is used. Suitable
particles for use in performing biological assays, e.g.,
immunoassays, are known in the art and are commercially available
from, e.g., Promega, Inc. (Madison, Wis.), Sigma-Aldrich (St.
Louis, Mo.), Magsphere, Inc. (Pasadena, Calif.), Dynal (now part of
Life Technologies, Carlsbad, Calif.). In one embodiment, magnetic
beads are retained in a receptacle using a magnetic force while
reagents are sequentially added to the receptacle and removed. In
one embodiment, a particle-based ELISA assay uses spectrally
discrete polystyrene beads to immobilize the capture antibody (or
other capture agent). For example, carboxy-coated microspheres
internally labeled with two fluorescent dyes that produce up to 100
different spectral addresses available from Bio-Rad Laboratories,
Inc. (Hercules, Calif.) may be used. Similar to some traditional
sandwich ELISA assays, each antibody-coupled bead captures analytes
that are detected with a biotinylated antibody and
phycoerythrin-conjugated streptavidin (SA-PE). For each capture
antibody-coupled bead, the reader simultaneously measures the
fluorescent signals of the bead's particular spectral address and
of the SA-PE. Each mean fluorescence intensity reading corresponds
to the average of the fluorescent signals from many (e.g., hundreds
to thousands) of antibody-coupled beads having a particular
spectral address. In the absence of cross-reactivity, each reading
can assess the concentration of multiple analytes that are detected
by spectrally distinct beads. The Bio-Plex Multiplex Suspension
Array System (Bio-rad) and Luminex's (Luminex; Austin, Tex.) flow
cytometer with carboxylate xMap.TM. microspheres (also Luminex) are
commercially available platforms that can be used to implement such
assays. Specific binding agents might be selected that bind to any
peptide, polypeptide, small molecule, or other analyte that is or
may be present in a biological sample in which complement
activation is to be assessed. Exemplary analytes include soluble
complement regulatory proteins (e.g., CFH), coagulation factors and
regulators, hormones, growth factors (e.g., vascular endothelial
growth factor), acute phase proteins (e.g., C-reactive protein),
cytokines, chemokines, cardiac markers (e.g., proteins that are
elevated in individuals who have suffered a heart attack), cancer
markers, etc.
[0074] In certain of the ELISA assays of the invention the capture
agent may be immobilized on a support by any suitable means,
provided that it remains capable of retaining the analyte following
immobilization. For example, a capture agent may be immobilized by
adsorption, covalent interaction, chelation, molecular recognition,
etc. In some embodiments a capture agent is immobilized via a
physical interaction between the capture agent and the support,
while in other embodiments the capture agent and support each
interact with a third entity that links the capture agent to the
support. For example, in some embodiments a first member of a
specific binding pair is linked to the support and a second member
of the specific binding pair is linked to the capture agent.
Binding of the members of the specific binding pair to each other
immobilizes the capture agent. In yet other embodiments a
bifunctional specific binding agent is used to attach the capture
agent to the support. The bifunctional specific binding agent
contains a first domain that links it to the support (or to a
moiety that is linked to the support) and a second domain that
links it to the capture agent (or to a moiety that is linked to the
capture agent). In some embodiments, capture agents are immobilized
on discrete spots on a support, e.g., within individual wells of a
microtiter plate. For example, a microtiter plate in which a
monoclonal antibody against C3a or a monoclonal antibody against
iC3b is adsorbed to individual wells may be used to perform certain
of the inventive assays. In other embodiments a polyclonal antibody
against C3 or C3d is adsorbed to individual wells and may be used
to perform certain of the inventive assays.
[0075] One of skill in the art will be aware that ELISA assays
typically include additional steps not necessarily described in
detail above, such as blocking, washing, preparing standard curves,
etc. Further details are provided in the Examples and in references
cited herein.
[0076] In some embodiments of the invention a standard curve for
use with samples comprising a particular matrix (e.g., serum,
plasma, vitreous, etc.) is prepared by "spiking" the matrix (which
may be a diluted matrix) with purified analyte (e.g., intact C3 or
iC3b) and measuring the signal obtained at different concentrations
of analyte. The concentrations may embrace the range of
concentrations that is desired to measure and/or that are expected
to be present in samples of interest (e.g., after appropriate
dilution). A background signal, e.g., the signal obtained from the
matrix alone may be subtracted from each value when preparing the
standard curve. The matrix used to generate the standard curve may
be diluted, in which case the background value subtracted may be
obtained at the same matrix dilution. For example, when preparing a
standard curve for samples containing 0.1% serum, a background
value due to 0.1% serum alone may be subtracted from the values
obtained using 0.1% serum spiked with an analyte. When using the
assay to measure complement activation on an actual sample
comprising that matrix, a background value obtained from a sample
of the diluent may be subtracted. Subtraction of the background
signal from a diluent sample can help minimize the effect of
non-specific binding of detection agent to the vessel or capture
agent. In some embodiments, a standard curve for an analyte of
interest is generated by spiking a matrix with the analyte of
interest, wherein the matrix has been depleted of endogenous
analyte.
[0077] In some embodiments of the invention the analyte is not
immobilized on a support prior to binding of a detection agent to
the analyte. For example, in some inventive assays an analyte of
interest and a specific binding agent bind in solution to form a
complex. Complexes containing the analyte and the specific binding
agent are subsequently detected, e.g., using turbidometry or
nephelometry. In some embodiments, complexes containing the analyte
and the specific binding agent are immobilized on a support prior
to detection. Unbound material is removed, and the support is
optionally subjected to one or more washing steps to remove
substances not bound to the specific binding agent.
[0078] In some embodiments a label-free detection method is used to
detect intact C3, iC3b, and/or total C3. For example, capture of an
analyte may be detected by detecting a change in mass, charge,
piezoelectricity, bending, surface plasmon resonance, or other
physical property, that occurs upon binding of the analyte to a
capture agent immobilized on a support. For example, cantilever
biosensors can be used (see, e.g., Fritz, J. Analyst,
133(7):855-63, 2008; Mutharasan, R., Methods Mol Biol. 504:73-82,
2009). The same capture agents as described for ELISA assays may be
used in these techniques. Other techniques that might be used in
various embodiments of the invention include radioimmunoassay,
immunodiffusion, nephelometry, electroimmunoassay, turbidometry,
time-resolved immunofluoremetric assay, Western blot,
immunoprecipitation, etc.
[0079] Without wishing to be bound by any theory, certain of the
inventive assays may show superior accuracy and/or reproducibility
relative to various other assays for complement and/or for
complement activation. In some embodiments, superior results are
achieved through use of a combination of selected capture and/or
detection agents, standard curves generated using the same matrix
as found in a biological sample of interest, and/or appropriate
sample dilutions.
III. KITS AND ASSAY SYSTEMS
[0080] The invention provides kits for use in performing one or
more inventive assays. Certain of the kits comprise at least one
specific binding agent that binds to intact C3. Certain of the kits
comprise at least one specific binding agent that binds to iC3b.
The kit may comprise any one or more of the specific binding agents
described herein. For example, in some embodiments a kit comprises
a polyclonal antibody that bind to C3 or C3d and a monoclonal
antibody that binds to C3a. Such kits are of use, e.g., to
determine the level of intact C3.
[0081] In some embodiments, a kit comprises at least one specific
binding agent that binds to total C3 and at least one specific
binding agent that binds to intact C3. In certain embodiments the
kits comprise at least three specific binding agents, wherein one
of the binding agents binds to a neoepitope of iC3b, one of the
binding agents bind to total C3, and one of the binding agents
binds to intact C3. In some embodiments a kit comprises a
polyclonal antibody that bind to C3, a monoclonal antibody that
binds to C3a, and a monoclonal antibody that binds to a neoepitope
of iC3b. In some embodiments a kit comprises a polyclonal antibody
that bind to C3d, a monoclonal antibody that binds to C3d, and a
monoclonal antibody that binds to C3a. In some embodiments the kit
comprises one or more receptacles, e.g., a microwell plate,
comprising a surface having a specific binding agent covalently or
noncovalently attached thereto, wherein the specific binding agent
serves as a capture agent for intact C3, iC3b, or total C3. For
example, the kit may comprise a microwell plate at least some of
whose wells are at least partly coated with an antibody that binds
to C3a, to C3d, to a neoepitope of iC3b, etc. In some embodiments
the kit comprises one or more populations of particles having a
specific binding agent attached thereto, wherein the specific
binding agent serves as a capture agent for intact C3, iC3b, or
total C3. For example, the kit may comprise particles having an
antibody attached covalently or noncovalently thereto, wherein the
antibody binds to C3a, to C3d, to a neoepitope of iC3b, to etc.
[0082] Any of the kits may further comprise at least one item
selected from the group consisting of: instructions for use of the
kit to assess complement activation; one or more native complement
components or cleavage products; human serum complement, optionally
characterized for levels of one or more complement components or
cleavage products; serum depleted of one or more complement
components or cleavage products; a detectably labeled secondary
antibody; an enzyme substrate; a buffer; a diluent; a protease
inhibitor; a complement inhibitor; and a support. In some
embodiments the kit comprises one or more proteins or polypeptides
selected from the group consisting of: C3 protein; C3d polypeptide;
C3a polypeptide, iC3b polypeptide, C3b polypeptide. The kit may
further comprise one or more specific binding agents that bind to
any native complement component or cleavage product.
[0083] Items in a kit will often be individually wrapped or
packaged in individual receptacles, which are provided together in
a larger container, e.g., a cardboard or styrofoam box. Often, the
reagents in the kit will have been tested and determined to be
suitable for use together in an inventive method. In some
embodiments reagents in the kit are suitable for use in a clinical
setting, e.g., to help guide diagnosis and/or treatment of
patients, as well as for research purposes. Such reagents or kits
may meet specified manufacturing and/or quality control criteria.
In some embodiment at least some of the items may be manufactured
according to good manufacturing practices.
[0084] The invention further provides assay systems comprising a
device useful for detecting a signal, e.g., a fluorescent or
luminescent or colorimetric signal, produced at least in part by a
detection agent. For example the device could comprise a
spectrometer, densitometer, or the like. The assay system comprises
an article, e.g., a microwell plate, comprising reagents useful for
performing an inventive assay. In some embodiments the assay system
comprises a plate reader. In some embodiments an assay system
comprises apparatus useful for performing an inventive assay in an
automated and/or high throughput manner, e.g., means for automating
one or more steps of the assay that might otherwise be performed
manually. Apparatus robots, liquid dispensers or other liquid
handling apparatus, automatic washing systems, means for moving
samples or receptacles from one position to another, etc. In some
embodiments, an assay system comprises a device, e.g., a computer
processor, for processing data acquired by the signal detecting
device, and, optionally, appropriate software for performing one or
more processing steps on the data. For example such processing
steps could include mathematical or statistical operations such as
subtracting background, obtaining an average value or a standard
deviation, obtaining a ratio, comparing values (e.g., comparing a
result obtained from a sample to a normal or reference value). In
some embodiments an assay system comprises means for storing data
and/or results from performing an inventive assay, means for
displaying or printing or otherwise outputting such data and/or
results, e.g., numerically or graphically. In some embodiments an
assay system comprises means for electronically transmitting data
or results of an inventive assay to another device, which may or
may not be physically connected with apparatus used to perform the
assay or process the data. The device may be located in a different
room, building, city, county, state, or country. The data or
results may be transmitted wirelessly. In some embodiments the data
or results are transmitted over a network, e.g., the Internet. In
some embodiments the invention provides a method comprising
receiving a sample, e.g., from a requestor; performing an inventive
assay on the sample; and providing data or results of the assay
(optionally electronically), e.g., to the requestor or the
requestor's designee. The requestor can be, e.g., a researcher or a
health care provider or a person or entity that acts on behalf of,
or employs, such individual. In some embodiments the invention
provides a method comprising furnishing a sample to a service
provider; and electronically receiving data or results of an
inventive assay performed on the sample. The service provider can
be any person or entity capable of performing an assay. In some
embodiments the method further comprises requesting the service
provider (optionally electronically) to measure complement
activation in the sample. In some embodiments the method further
comprises obtaining the sample from a subject.
[0085] In some embodiments the invention provides a method
comprising performing an inventive assay using an assay system
described herein or a component thereof.
IV. APPLICATIONS
[0086] The inventive methods and kits may be used in a number of
applications. For example, in certain embodiments an assay of this
invention may be used to assess the level of intact C3 or iC3b in a
subject and/or to assess the extent to which an agent, e.g., a
complement inhibitor, or a material that has the potential to
activate complement, affects the level of intact C3 or iC3b in vivo
or in vitro. In some embodiments, an assay of this invention may be
used to assess the extent to which complement is activated and/or
to assess the extent to which an agent affects, e.g. increases or
decreases, complement activation in vivo or in vitro. In some
embodiments, an inventive method is performed on a biological
sample obtained from a subject. In some embodiments, no more than
0.1%, 0.5%, 1%, or 5% of the proteins in the sample by dry weight
are complement components. In some embodiments, intact C3 or iC3b
and/or complement activation are measured using an assay
composition that comprises at least some substantially purified
complement components. For example, a sample may contain one or
more proteins, wherein at least 50% of the proteins by dry weight
are complement components. In some embodiments an assay composition
contains C3 and all intact complement components that lie
"upstream" of C3 in one or more complement activation pathways. The
assay composition may further comprise an agent that activates
complement. For example, the assay composition may comprise IgM to
activate the classical pathway-lipopolysaccharide to activate the
alternative pathway, mannan (to activate the mannose-binding lectin
portion of the lectin pathway). The ability of an agent to inhibit
such activation may be assessed using an inventive assay. One may
select appropriate assay conditions (e.g., temperature, pH, etc.)
if it is desired to selectively activate and/or assess one of the
pathways. Optionally the level of intact C3, iC3b and/or the extent
of complement activation is compared with a suitable reference
value. The reference value may be, e.g., a value measured from a
sample that lacks one or more complement components, a value
measured from a sample that lacks an agent that activates
complement, a value measured from a sample obtained from a healthy
individual, etc. In some embodiments an inventive assay is used to
assess the effect of an agent or condition on the synthesis or
secretion of C3. The assay could be used to measure intact C3
produced by, e.g., cultured hepatocytes, retinal pigment epithelial
(RPE) cells, or any other cells that may synthesize C3. In some
embodiments an inventive assay is used to screen for agents that
inhibit or enhance synthesis and/or secretion of C3 by cells, e.g.,
hepatocytes or RPE cells. Such agents would be of use to modulate
the complement system, e.g., for therapeutic purposes. In certain
aspects, a method of the invention is of use for assessing
proteolytic activity towards a complement component, e.g.,
proteolytic activity associated with a complement activating
substance or condition.
[0087] In some embodiments, intact C3, iC3b, total C3, and/or
complement activation is assessed in a blood, plasma, or serum
sample obtained from a subject. In other embodiments, an inventive
assay is used to assess the level of C3, iC3b, total C3, and/or
complement activation in a sample of aqueous humor or vitreous
humor. In some embodiments, intact C3, iC3b, total C3, and/or
complement activation levels in the respiratory tract can be
measured in a sputum sample. In some embodiments, intact C3, iC3b,
total C3, and/or complement activation levels in a joint space can
be measured in a sample of synovial fluid. In some embodiments,
intact C3, iC3b, total C3, and/or complement activation levels in
the CNS can be measured in a sample of CSF.
[0088] In some embodiments, an inventive assay is used to assess
the effect on complement activation in vivo of an agent that has
been administered to a subject. The agent is administered to the
subject, and a sample is subsequently obtained. The extent of
complement activation in the sample is assessed. Optionally the
extent of complement activation is compared with a suitable
reference value. The reference value may be, e.g., a value measured
from a sample obtained from the subject prior to administration of
the agent, an average value measured from samples obtained from a
group of "healthy" individuals, a value that represents a desired
extent of complement activation, etc. In some embodiments the agent
whose effect on complement activation is assessed is a complement
inhibitor. The complement inhibitor may be, e.g., a compound that
inhibits at least one complement activation pathway as assessed
using, e.g., any of the standard assays for complement activation
described herein or known in the art. In some embodiments a
complement inhibitor acts on a complement component or cleavage
product, e.g., selected from C1, C3, C3b, factor B, or factor D. In
some embodiments a complement inhibitor acts at or above the level
of C3 convertase in a complement activation pathway, e.g., the
complement inhibitor does not directly act on C5, C5 convertase, or
a molecule that participates in one or more steps of complement
activation that occurs in a complement activation pathway
subsequent to formation of C5 convertase. In some embodiments, an
inventive assay is used to assess the effect of a complement
inhibitor on levels of intact C3. In some embodiments, an inventive
assay is used to assess the effect of a complement inhibitor on
levels of iC3b. For example, a first sample is obtained from a
subject before administration of a complement inhibitor and a
second sample is obtained after administration of the complement
inhibitor. Optionally the subject is exposed to a complement
activating stimulus prior to obtaining the second sample. In some
embodiments, levels of intact C3 are measured in the samples. If
the level of intact C3 in the second sample is greater than a
control value (e.g., the value that would be expected had the
complement inhibitor not been administered), it can be concluded
that the complement inhibitor inhibited complement activation and
thus inhibited consumption of intact C3. In some embodiments,
levels of iC3b are measured in the samples. If the level of iC3b in
the second sample is lower than a control value (e.g., the value
that would be expected had the complement inhibitor not been
administered), it can be concluded that the complement inhibitor
inhibited complement activation and thus inhibited generation of
iC3b.
[0089] Examples of agents that can inhibit one or more complement
activation pathways include: antibodies that specifically bind to a
complement component such as C1 (or a subunit thereof), C3, C5
(e.g., a humanized monoclonal anti-C5 antibody such as 5G1.1-scFv,
Pexelizumab, Eculizumab), factor B (e.g., TA106 a monoclonal
antibody fragment against Factor B, or humanized versions thereof),
or factor D (e.g., TNX-234, a humanized monoclonal antibody that
binds Factor D); soluble complement receptors or molecules
comprising them (e.g., soluble complement receptor 1 (sCR1) also
known as TP10); C1-INH; certain small molecules; certain peptides,
certain polypeptides comprising at least a portion of a mammalian,
e.g., human, complement regulatory protein such as CFH, CFI, CR1,
decay accelerating factor (DAF; CD55), membrane cofactor protein
(MCP; CD46), CD59, C4 bp, or CRIT (e.g., the polypeptide known as
MLN2222--a fusion protein derived from DAF and MCP (U.S. Pat. No.
5,679,546); viral complement control proteins and viral complement
interfering proteins (see, e.g., U.S. Ser. No. 11/612,751);
naturally occurring complement inhibitors produced by bacteria,
parasites, insects; and aptamers that bind to a complement
component, etc. In some embodiments the complement inhibitor is
compstatin or an analog thereof. Exemplary compstatin analogs are
disclosed in, e.g., WO2004/026328, U.S. Ser. No. 11/605,182; and/or
U.S. Ser. No. 11/544,389. In some embodiments the compstatin analog
comprises a compound having a sequence set forth in Table 1 (e.g.,
SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 34), e.g., cyclized via a
bond between the cysteine residues, e.g., a disulfide bond. See
Ricklin, D., et al. "Complement-targeted Therapeutics", Nature
Biotechnology, 25(11):1265-75, 2007, for discussion of complement
inhibitors that are or have been in preclinical or clinical
development for various disorders.
TABLE-US-00001 TABLE 1 SEQ Activity ID over Peptide Sequence NO:
compstatin Compstatin H-ICVVQDWGHHRCT-CONH2 8 * Ac-compstatin
Ac-ICVVQDWGHHRCT-CONH2 9 3xmore Ac-V4Y/H9A Ac-ICVYQDWGAHRCT-CONH2
10 14xmore Ac-V4W/H9A-OH Ac-ICVWQDWGAHRCT-COOH 11 27xmore
Ac-V4W/H9A Ac-ICVWQDWGAHRCT-CONH2 12 45xmore Ac-V4W/H9A/T13dT-OH
Ac-ICVWQDWGAHRCdT-COOH 13 55xmore Ac-V4(2-Nal)/H9A
Ac-ICV(2-Nal)QDWGAHRCT-CONH2 14 99xmore Ac V4(2-Nal)/H9A-OH
Ac-ICV(2-Nal)QDWGAHRCT-COOH 15 38xmore Ac V4(1-Nal)/H9A-OH
Ac-ICV(1-Nal)QDWGAHRCT-COOH 16 30xmore Ac-V42Igl/H9A
Ac-ICV(2-Igl)QDWGAHRCT-CONH2 17 39xmore Ac-V42Igl/H9A-OH
Ac-ICV(2-Igl)QDWGAHRCT-COOH 18 37xmore Ac-V4Dht/H9A-OH
Ac-ICVDhtQDWGAHRCT-COOH 19 5xmore Ac-V4(Bpa)/H9A-OH
Ac-ICV(Bpa)QDWGAHRCT-COOH 20 49xmore Ac-V4(Bpa)/H9A
Ac-ICV(Bpa)QDWGAHRCT-CONH2 21 86xmore Ac-V4(Bta)/H9A-OH
Ac-ICV(Bta)QDWGAHRCT-COOH 22 65xmore Ac-V4(Bta)/H9A
Ac-ICV(Bta)QDWGAHRCT-CONH2 23 64xmore Ac-V4W/H9(2-Abu)
Ac-ICVWQDWG(2-Abu)HRCT-CONH2 24 64xmore +G/V4W/H9A + AN-OH
H-GICVWQDWGAHRCTAN-COOH 25 38xmore Ac-V4(5fW)/H9A
Ac-ICV(5fW)QDWGAHRCT-CONH.sub.2 26 31xmore Ac-V4(5-MeW)/H9A
Ac-ICV(5-methyl-W)QDWGAHRCT-CONH.sub.2 27 67xmore
Ac-V4(1MeW)/W7(5fW)/H9A Ac-ICV(1-methyl-W)QD(5fW)GAHRCT- 28
264xmore CONH.sub.2 Ac-V4W/W7(5fW)/H9A
Ac-ICVWQD(5fW)GAHRCT-CONH.sub.2 29 121xmore Ac-V4(5fW)/W7(5fW)/H9A
Ac-ICV(5fW)QD(5fW)GAHRCT-CONH.sub.2 30 161xmore
Ac-V4(5-MeW)/W7(5fW)H9A Ac-ICV(5-methyl-W)QD(5fW)GAHRCT- 31 NA
CONH.sub.2 Ac-V4(1-MeW)/H9A Ac-ICV(1-methyl-W)QDWGAHRCT-CONH.sub.2
32 264xmore +G/V4(6fW)/W7(6fW)H9A + N-
H-GICV(6fW)QD(6fW)GAHRCTN-COOH 33 126xmore OH Ac-V4(1-formyl-W)/H9A
Ac-ICV(1-formyl-W)QDWGAHRCT-CONH.sub.2 34 264xmore
Ac-V4(5-methoxy-W)/H9A Ac-ICV(1-methyoxy-W)QDWGAHRCT- 35 76xmore
CONH.sub.2 G/V4(5f-W)/W7(5fW)/H9A + N-
H-GICV(5fW)QD(5fW)GAHRCTN-COOH 36 112xmore OH N/A = not
available
[0090] In some embodiments an inventive assay is used to assess a
complement inhibitor or a complement activator in vitro. The
complement activator can be any substance that activates complement
as assessed using, e.g., any of the standard assays for complement
activation described herein or known in the art. Examples of agents
known to activate complement are: bacterial cell wall components
such as LPS or carbohydrates, antigen-antibody complexes, etc. In
some embodiments the agent is an agent being tested to determine
its effect on complement activation. In some embodiments the agent
is a therapeutic agent whose mechanism of action is at least in
part mediated by complement. The agent may be an antibody that
activates complement on target cells such as cancer cells, immune
system cells that play a role in autoimmune disease, or pathogens.
Examples of such agents include antibodies that bind to various
cell surface molecules such as CD20, EGFR, HER2, or other growth
factor receptors. Specific examples include rituximab, cetuximab,
trastuzumab, and alemtuzumab. In some embodiments, measurements are
made on samples obtained at multiple time points, optionally
starting before administration of an agent, thereby providing an
indication of the extent to which the agent modulates complement
activation and the time course and duration of the modulation.
[0091] In some embodiments of the invention, information obtained
from an inventive assay is used to provide prognostic, diagnostic,
or therapeutic information relating to a subject. "Prognostic
information" can include information regarding the likelihood that
a subject will develop a disease or condition and/or information
regarding the possible outcome, time course, or progression of a
disease or condition (e.g., with or without therapy), etc.
"Diagnostic information" can include information regarding the
likelihood that a subject has a disease or condition and/or
information about the particular features of such disease or
condition as manifested in the subject. "Therapeutic information"
can include information regarding a suitable treatment or
therapeutic regimen for the subject. For example, therapeutic
information can include whether to administer a therapeutic agent,
which agent or class of agents is appropriate, which dose and
dosing schedule to select, whether and when to retreat, whether to
cease therapy, etc. In some embodiments, e.g., embodiments in which
the therapeutic agent is a complement inhibitor, the disorder is a
complement-mediated disorder, i.e., one in whose development,
progression, or manifestation(s) complement activation plays a
role. In some embodiments the condition is associated with
complement deficiency.
[0092] In some embodiments the level of intact C3, iC3b, total C3,
or complement activation in a sample comprising, e.g., serum,
plasma, or blood from a subject are determined, and the result is
used to provide prognostic, diagnostic, or therapeutic information.
For example, results of an inventive assay may be used, optionally
together with information regarding the subject's genotype with
respect to one or more alleles or polymorphisms that have been
associated with increased or decreased risk of developing a disease
and/or with increased or decreased risk of progression of a
disease, in order to provide prognostic, diagnostic, or therapeutic
information. In some embodiments, the genotype is obtained with
respect to particular alleles of one or more complement components
or complement regulatory proteins. For example, in some embodiments
the genotype is determined with respect to a polymorphic site in a
gene that encodes a protein selected from the group consisting of:
complement factor H (CFH), complement proteins C2, C3, factor B,
C7, complement factor I, a CFH-like protein (e.g., CFHRJ, CFHRJ,
CFHR3, CFHR4, and CFHR5), and MBL-2.
[0093] In some embodiments, the disorder is an ocular disorder
(e.g., a disease that only or primarily affects the eye). Exemplary
ocular disorders are macular degeneration, e.g., age-related
macular degeneration (AMD), diabetic retinopathy, glaucoma, and
uveitis. AMD is an ocular disorder that is characterized by
degenerative changes in the macula, the area of the retina that
provides high visual acuity and contains a dense concentration of
cones, the photoreceptors that are responsible for color vision.
AMD causes a progressive loss in central vision and is the most
common cause of functional blindness in developed countries for
those over 50 years of age. AMD has been classified into dry
(non-exudative) and wet (exudative) forms. The wet form, which
affects about 10-15% of those with AMD, is characterized by
progressive growth of new blood vessels (neovascularization) in the
back of the eye. Pathologic neovascularization can cause rapid
deterioration in vision as a consequence of fluid leakage and, in
the longer term, retinal fibrosis and scarring. The majority of
individuals with AMD suffer from the dry form of the disorder,
which is characterized by atrophy and dysfunction or retinal
tissues but lacks the ocular neovascularization present in the wet
form. Dry AMD can cause serious vision loss, and persons with dry
AMD are at increased risk of progressing to the wet form. In the
Age-Related Eye Disease Study (AREDS), individuals were classified
into AREDS category 1 (No AMD--no or few small drusen<63 microns
in diameter), AREDS category 2 (consisting of a combination
multiple small drusen (<63 microns in diameter), few
intermediate drusen (63-124 microns in diameter), or RPE
abnormalities), AREDS category 3 (consisting of extensive
intermediate drusen, at least one large drusen (>125 microns in
diameter)), or GA not involving the center of the fovea), or AREDS
4 (GA involving the center of the fovea or exudative AMD). Further
information regarding AMD is found in the following: Jager, R., et
al., N. Engl. J. Med.: 358:2606-17, 2008, and other references
cited herein. See also, American Academy of Ophthalmology Retina
Panel. Preferred Practice Pattern.RTM. Guidelines. Age-Related
Macular Degeneration. San Francisco, Calif.: American Academy of
Ophthalmology; 2008, available at www.aao.org./ppp.
[0094] A variety of clinical and imaging modalities may be used to
assess subjects at risk of or suffering from AMD. Visual acuity can
be measured using, for example, a Snellen chart, a Bailey-Lovie
chart, a decimal progression chart, a Freiburg visual acuity test,
a measurement of minimum angle of resolution (MAR) etc.
Metamorphopsia (visual distortion) may be measured using an Amsler
chart. Contrast sensitivity may be measured using a Pelli-Robson
chart. Diagnostic studies include, but are not limited to, standard
ophthalmologic examination of the fundus, stereo biomicroscopic
examination of the macula, intravenous fundus fluorescein
angiography, fundus photography, indocyanine green
video-angiography, optical coherence tomography, autofluorescence
studies, and other imaging techniques. An individual who would be
recognized as having AMD by one of skill in the art based on
appropriate physical findings, imaging tests, and/or other
appropriate criteria is considered to have "clinically evident
AMD".
[0095] The present invention provides, in some aspects, new
approaches for assessing subjects at risk or suffering from AMD.
Surprisingly, it was found that individuals that had been diagnosed
with AMD had significantly higher serum levels of intact C3 than
control individuals over 60 years of age not diagnosed with AMD
(see Example 9). Notably, the levels of intact C3 found in the two
groups of samples (from individuals with or without diagnosed AMD)
fell into two non-overlapping ranges, with the exception of one
sample in the control (non-AMD) group (which might have represented
an undiagnosed case of AMD or a subject developing but not yet
having clinically evident AMD). The invention encompasses the
recognition that levels of intact C3 (e.g., blood, serum, or plasma
levels) can be used as a biomarker for AMD. The invention provides
methods of using the level of intact C3 in a biological sample,
e.g., blood, serum, or plasma, to provide prognostic, diagnostic,
or therapeutic information relating to AMD. In some embodiments of
the invention, the level of intact C3 is used to assess whether a
subject has or is at increased risk of developing clinically
evident AMD. By "at increased risk of developing clinically evident
AMD" is meant that the subject has a greater likelihood of
developing clinically evident AMD than the average likelihood that
an individual of the same age but not having an increased level of
intact C3 has of developing clinically evident AMD. In some
embodiments the subject is at increased risk of developing
clinically evident AMD within the subsequent 6 months, 1 year, 2
years, or 5 years. The subject may be in the process of developing
AMD, i.e., their condition is progressing towards clinically
evident AMD.
[0096] The invention provides a method for assessing whether a
subject has, or is at increased risk of developing, age-related
macular degeneration (AMD), or is at increased risk of progressing
from early AMD to more advanced AMD, comprising detecting intact C3
in a sample comprising serum, plasma, or whole blood from the
subject, wherein a level of intact C3 that is greater than a
control level indicates that the individual has or is at increased
risk of developing AMD, or is at increased risk of progressing from
early AMD to more advanced AMD. In some embodiments the control
level is an average level found in a control population composed of
individuals not having AMD. In some embodiments the control
population is composed of individuals at least 60 years of age not
having AMD. In some embodiments the control population is composed
of individuals not having AMD who are within 5 years, or within 10
years, of the age of the subject.
[0097] The invention also provides a related method for assessing
whether a subject has, or is at increased risk of developing,
age-related macular degeneration (AMD), or is at increased risk of
progressing from early AMD to more advanced AMD, comprising
detecting intact C3 in a sample comprising serum, plasma, or whole
blood from the subject, wherein a level of intact C3 that is within
a range associated with increased likelihood of having or
developing AMD or associated with increased risk of progressing
from early AMD to more advanced AMD, indicates that the individual
has or is at increased risk of developing AMD, or is at increased
risk of progressing from early AMD to more advanced AMD.
[0098] In some embodiments the method of assessing the level of
intact C3 comprises detecting intact C3 using an ELISA assay. In
some embodiments the method comprises capturing intact C3 using a
monoclonal antibody that binds to C3a and detecting captured intact
C3 using a polyclonal antibody that binds to C3. In some
embodiments the method comprises capturing intact C3 using a
monoclonal antibody that binds to C3a and detecting intact C3 using
a polyclonal antibody that binds to C3d and does not significantly
recognize C3 epitopes outside C3d. In some embodiments the
monoclonal antibody is mouse anti-human C3a/C3 antibody #HM2075
(Cell Sciences) or an antibody that binds to the same epitope.
[0099] In some embodiments the subject has not been diagnosed with
AMD, and an increased level of intact C3 relative to a control
level indicates that the subject is developing AMD, e.g., the
subject is progressing towards clinically evident AMD. In some
embodiments the subject has not been diagnosed with AMD, and an
increased level of intact C3 indicates that the subject is at
increased risk of manifesting clinically evident AMD, e.g., within
the following 6 months, 1 year, 2 years, 5 years, or 10 years,
relative to the likelihood that a subject not having an increased
level of intact C3 would have.
[0100] In some embodiments, the individual has early AMD, and a
level of intact C3 that is greater than a control level indicates
that the individual is at increased risk of progressing from early
AMD to more advanced AMD, e.g., within the following 6 months, 1
year, 2 years, 5 years, or 10 years, relative to the likelihood
that a subject not having an increased level of intact C3 would
have of progressing. In some embodiments, the individual has early
AMD, and a level of intact C3 that is greater than a control level
indicates that the individual is progressing to more advanced AMD.
In some embodiments of the invention, the level of intact C3 is
used to assess whether a subject with early AMD (e.g., AREDS 2) is
at increased risk of progressing to more advanced AMD such as AMD
with geographic atrophy or wet AMD, i.e., to assess whether a
subject with early AMD has a greater likelihood of developing more
advanced AMD than the average likelihood that an individual with
early AMD has of developing advanced AMD. In some embodiments of
the invention, the level of intact C3 is used to assess whether a
subject with GA not involving the fovea is at increased risk of
progressing to GA involving the fovea, i.e., to assess whether a
subject with GA not involving has a greater likelihood of
progressing to GA involving the fovea than the average likelihood
that an individual with GA not involving the fovea has of
progressing to GA involving the fovea. In some embodiments, the
increased level is greater than the control level by a
statistically significant amount. In some embodiments, the
increased level is at least 1.5 times the average control level. In
some embodiments, the increased level is at least 2 times the
average control level. In some embodiments the likelihood is
increased by a factor of at least 1.2, at least 1.5, at least 2, at
least 2.5, at least 3, at least 4, at least 5, or more.
[0101] In some embodiments, intact C3 levels are monitored at
various times (e.g., annually), e.g., in individuals over age 50.
If the level of intact C3 exceeds a predetermined value and/or
exhibits a significant increase as compared with a previously
determined level, the individual is started on appropriate therapy,
e.g., with a potent compstatin analog. In some embodiments, intact
C3 levels are monitored over time in individuals with early AMD. If
the level of intact C3 exceeds a predetermined value and/or
exhibits a significant increase as compared with a previously
determined level, the individual is started on appropriate therapy,
e.g., with a potent compstatin analog.
[0102] The invention also provides a method for assessing whether a
subject has or is at increased risk of developing age-related
macular degeneration (AMD) comprising determining the level of one
or more proteins in a sample comprising serum, plasma, or whole
blood from the subject using a monoclonal antibody that binds to
C3a/C3, wherein a level of said one or more proteins that is
greater than a control level indicates that the individual has or
is at increased risk of developing AMD. In some embodiments, the
protein(s) is/are detected using an ELISA assay. In some
embodiments the method comprises capturing protein(s) using a
monoclonal antibody that binds to C3a/C3 and detecting captured
intact C3 using a polyclonal antibody that binds to C3. In some
embodiments the method comprises capturing protein(s) using a
monoclonal antibody that binds to C3a/C3 and detecting intact C3
using a polyclonal antibody that binds to C3d and does not
significantly recognize C3 epitopes outside C3d. In some
embodiments the monoclonal antibody is mouse anti-human C3a/C3
antibody #HM2075 (Cell Sciences) or an antibody that binds to the
same epitope.
[0103] In some embodiments the subject has not been diagnosed with
AMD, and an increased level of proteins captured using the antibody
relative to a control level indicates that the subject is
developing AMD, e.g., the subject is progressing towards clinically
evident AMD. In some embodiments the subject has not been diagnosed
with AMD, and an increased level of proteins captured using the
antibody indicates that the subject is at increased risk of
manifesting clinically evident AMD, e.g., within the following 6
months, 1 year, 2 years, 5 years, or 10 years, relative to the
likelihood that a subject not having an increased level of intact
C3 would have.
[0104] In some embodiments, the individual has early AMD, and a
level of proteins captured using the antibody that is greater than
a control level indicates that the individual is at increased risk
of progressing from early AMD to more advanced AMD, e.g., within
the following 6 months, 1 year, 2 years, 5 years, or 10 years,
relative to the likelihood that a subject not having an increased
level of proteins captured using the antibody would have of
progressing. In some embodiments, the individual has early AMD, and
a level of proteins captured using the antibody that is greater
than a control level indicates that the individual is progressing
to more advanced AMD. In some embodiments of the invention, the
level of proteins captured using the antibody is used to assess
whether a subject with early AMD (e.g., AREDS 2) is at increased
risk of progressing to more advanced AMD such as AMD with
geographic atrophy or wet AMD, i.e., to assess whether a subject
with early AMD has a greater likelihood of developing more advanced
AMD than the average likelihood that an individual with early AMD
has of developing advanced AMD. In some embodiments of the
invention, the level of proteins captured using the antibody is
used to assess whether a subject with GA not involving the fovea is
at increased risk of progressing to GA involving the fovea, i.e.,
to assess whether a subject with GA not involving has a greater
likelihood of progressing to GA involving the fovea than the
average likelihood that an individual with GA not involving the
fovea has of progressing to GA involving the fovea. In some
embodiments, the increased level is greater than the control level
by a statistically significant amount. In some embodiments, the
increased level is at least 1.5 times the average control level. In
some embodiments, the increased level is at least 2 times the
average control level. In some embodiments the likelihood is
increased by a factor of at least 1.2, at least 1.5, at least 2, at
least 2.5, at least 3, at least 4, at least 5, or more.
[0105] In some embodiments, the level of proteins captured using
the antibody is monitored at various times (e.g., annually), e.g.,
in individuals over age 50. If the level exceeds a predetermined
value and/or exhibits a significant increase as compared with a
previously determined level, the individual is started on
appropriate therapy, e.g., with a potent compstatin analog. In some
embodiments, levels are monitored over time in individuals with
early AMD. If the level exceeds a predetermined value and/or
exhibits a significant increase as compared with a previously
determined level, the individual is started on appropriate therapy,
e.g., with a potent compstatin analog.
[0106] Results of an inventive assay may be used, optionally in
conjunction with one or more other assays and/or information
obtained from one or more clinical or laboratory evaluations. to
provide prognostic, diagnostic, or therapeutic information. For
example, results of an inventive assay, optionally together with
information obtained from an ophthalmoscopic examination, other
physical examination, genetic evaluation, risk factor evaluation
(e.g., diet, smoking status, age), family history, etc., can be
used to provide prognostic, diagnostic, or therapeutic information.
In some embodiments, results of an inventive assay may be used,
optionally together with information regarding the subject's
genotype with respect to one or more alleles or polymorphisms that
have been associated with increased or decreased risk of developing
a disease and/or with increased or decreased risk of progression of
a disease. In some embodiments, the genotype is obtained with
respect to particular alleles of one or more complement components
or complement regulatory proteins. For example, in some embodiments
the genotype is determined with respect to a polymorphic site in a
gene that encodes a protein selected from the group consisting of:
complement factor H (CFH), complement proteins C2, C3, factor B,
C7, complement factor I, a CFH-like protein (e.g., CFHR1, CFHR1,
CFHR3, CFHR4, and CFHR5), and MBL-2.
[0107] Genetic studies have identified strong associations between
alleles of a number of complement components and complement
regulatory proteins and the risk of developing AMD. See, e.g., Li,
M, et al., Nat Genet, 38:1049-54, 2006 (see, Tables 1 and 2); Gold,
B., et al., Nat. Genet, 38: 458-62, 2006; Dinu, V., et al., Genetic
Epidemiology, 31: 224-237, 2007 (see, e.g., Tables 3 and 5), Yates,
J. R. W., N. Engl. J. Med., 357: 19-27, 2007 (see, e.g., Tables 2
and 3), Francis, P., et al., PLoS ONE. November 28; 2(11):e1197,
2007 (see, e.g., Tables 1 and 2 therein), Fagerness J A, et al.,
Eur J Hum Genet., 17(1):100-4, 2009; Bergeron-Sawitzke J, et al.,
Eur J Hum Genet. 2009 Mar. 4. [Epub ahead of print]. Polymorphisms
linked to altered AMD susceptibility include rs1061170, rs1047286,
rs2230199, rs120862610, rs9332739, rs547154, rs4151667, rs641153,
rs41015361, rs33682798, rs10490924, and rs1045216. Polymorphic
sites in age-related maculopathy susceptibility (ARMS2) and/or HtrA
serine peptidase 1 (HTRA1) loci are also associated with altered,
e.g., increased, susceptibility to AMD. In some embodiments,
results of an inventive assay are used, optionally together with
genotype information with respect to one or more alleles that are
associated with increased or decreased risk of developing AMD, to
provide prognostic, diagnostic, or therapeutic information. In some
embodiments, an increased level of complement activation as
assessed using an inventive assay, together with presence of one or
more risk alleles for AMD indicates that the subject has an
increased likelihood of developing AMD or of progressing rapidly
from an early form of dry AMD (e.g., AREDS 2) to a more advanced
form such as geographic atrophy (GA) or of progressing from dry AMD
to wet AMD or of a subject with wet AMD developing GA, relative to
the likelihood that a subject having the risk allele but not having
an increased level of intact C3. In some embodiments, an increased
level of intact C3, together with presence of one or more risk
alleles for AMD indicates that the subject has an increased
likelihood of developing AMD or of progressing rapidly from an
early form of dry AMD (e.g., AREDS 2) to a more advanced form such
as geographic atrophy (GA), relative to the likelihood that a
subject having the risk allele but not having an increased level of
intact C3. In some embodiments the methods allow one to identify
subjects who are likely to progress to clinically evident AMD
within a subsequent time period such as the 6 months, 1 year, or 2
years after the assay was performed. In some embodiments the
methods allow one to identify subjects who are not likely to
progress to clinically evident AMD within a subsequent time period
such as the 6 months, 1 year, or 2 years after the assay was
performed. Based at least in part on such information, an
appropriate therapy, e.g., an appropriate complement inhibitor
therapy, e.g., therapy with a potent compstatin analog, may be
recommended to a subject. See, e.g., U.S. Ser. No. 11/544,389 and
PCT/US08/78593. See also PCT/US07/01649. Based at least in part on
such information, a health care provider may select complement
inhibitor therapy for a subject. The subject may, for example, have
drusen whose phenotype indicates an increased likelihood that the
subject will develop AMD within 5 years time (versus the likelihood
that an appropriately matched subject without drusen would develop
AMD within 5 years). If the subject additionally has an elevated
level of intact C3, therapy is initiated. In other embodiments the
subject may have early dry AMD that has not progressed to GA. If
the subject additionally has an elevated level of intact C3,
therapy is initiated. On the other hand, if the subject does not
have an elevated level of intact C3, the subject can continue being
monitored without initiating therapy. In some embodiments an assay
of the invention is used, optionally together with genotype,
clinical, imaging, or other data, to decide when to initiate
complement inhibitor therapy. Samples (e.g., blood or anterior
chamber fluid) are obtained at various time points (e.g.,
approximately monthly, every 6-8 weeks, etc.) from a subject at
risk of AMD or of progressing from early to intermediate or from
intermediate to advanced AMD, and an inventive assay is performed.
In some embodiments, if the level of intact C3 in the subject's
blood rises above a specified value, complement inhibitor therapy
is initiated. In some embodiments, if the level of complement
activation rises significantly above a specified value, complement
inhibitor therapy is initiated. The specified value may be, e.g.,
the average value found in a population of subjects not suffering
from AMD (e.g., subjects above 60 years of age), or between 1 and 5
times such value. In some embodiments, a complement inhibitor is
administered to a subject suffering from AMD, e.g., early,
intermediate, or advanced AMD (wet AMD or GA involving the fovea)
in one or both eyes. Samples (e.g., blood or anterior chamber
fluid) are obtained at various time points (e.g., approximately
monthly, every 6-8 weeks, etc.) and an inventive assay performed.
If the level of complement activation rises above a specified
value, the subject is retreated with the complement inhibitor. The
specified value may be, e.g., the average value found in a
population of subjects not suffering from AMD, or a fraction of
such value, e.g., a fraction between 1/20 and 1.
[0108] In some embodiments an inventive method is used to provide
prognostic, diagnostic or therapeutic information, wherein the
disorder is a renal disease (e.g., a disease that exclusively or
primarily affects the kidney) or a systemic disease that has renal
manifestations, e.g., glomeruloephritis, e.g., glomerulonephritis
with C3 deposition such as membranoproliferative glomerulonephritis
(MPGN) or atypical haemolytic uraemic syndrome (aHUS).
[0109] In some embodiments, an inventive method is used to provide
prognostic, diagnostic or therapeutic information, wherein the
disorder is one that affects the cardiovascular system, such as
atherosclerosis or restenosis (e.g., following an intervention such
as angioplasty, balloon dilation, laser ablation, or stent
placement). In some embodiments an inventive assay is used to
monitor complement activation over time in a subject who suffers
from or is at risk of a disorder affecting the cardiovascular
system, and a complement modulating agent is administered so as to
keep the level of complement activation within desired values. For
example, the subject may have suffered a myocardial infarction, may
have elevated cholesterol (e.g., LDL cholesterol) and/or
triglycerides, a family history of cardiovascular disease, etc. The
desired value may be, e.g., the average value found in a population
of subjects, e.g., a population of subjects not suffering from a
cardiovascular disorder, or a fraction of such value, e.g., a
fraction between 5% and 99% of such value.
[0110] In some embodiments the subject has suffered an injury,
e.g., a serious physical injury such as an open wound, blunt
injury, or major burns. In some embodiments of the invention the
subject suffers from traumatic brain injury or spinal cord injury.
In some embodiments the subject has suffered an injury sufficiently
severe to warrant admission to hospital or to an intensive care
unit. In some embodiments the subject has an injury severity score
(ISS) of at least 9. In some embodiments the subject has an injury
severity score (ISS) of at least 15. The Injury Severity Score
(ISS) is an anatomical scoring system that provides an overall
score for patients with multiple injuries Baker S P et al, "The
Injury Severity Score: a method for describing patients with
multiple injuries and evaluating emergency care", J Trauma
14:187-196, 1974. The ISS score is the most widely used anatomical
scoring system for injury and correlates linearly with mortality,
morbidity, hospital stay and other measures of severity. In some
embodiments an inventive assay is used to monitor complement
activation over time in a subject who has recently suffered severe
physical injury and a complement modulating agent is administered
so as to keep the level of complement activation within desired
values. In some embodiments "recently" refers to within the
preceding 24, 48, 72, 96, or 120 hours. In some embodiments
"recently" refers to within the preceding 1, 2, 3, 4, 5, 6, 7, 10,
14, 21, or 28 days. The desired value may be, e.g., the average
value found in a population of subjects not suffering from severe
injury, or a fraction thereof, e.g., a fraction between 5% and 99%
of such value. In some embodiments the desired value is greater
than the average value found in subjects not suffering from severe
injury. For example, the desired value may be between 0.5 and 5
times such value.
[0111] In some embodiments the subject has recently suffered an
event associated with ischemia (e.g., an ischemic stroke or a heart
attack). In some embodiments the subject is at risk of or suffers
from ischemia/reperfusion injury. In some embodiments the subject
has suffered a hemorrhage.
[0112] In some embodiments the subject has experienced an adverse
reaction to a drug or a reaction to an environmental antigen, food,
toxin, venom, etc.
[0113] In some embodiments of the invention the disorder is
selected from the group consisting of alopecia areata, anklosing
spondylitis, antiphospholipid syndrome, asthma, autoimmune
Addison's disease, autoimmune hemolytic anemia, autoimmune
hepatitis, autoimmune inner ear disease, autoimmune
lymphoproliferative syndrome (alps), autoimmune thrombocytopenic
purpura (ATP), Behcet's disease, bullous pemphigoid,
cardiomyopathy, celiac sprue, chronic fatigue syndrome immune
deficiency, syndrome (CFIDS), chronic inflammatory demyelinating
polyneuropathy, chronic obstructive pulmonary disease (COPD),
cicatricial pemphigoid, cold agglutinin disease, Crest syndrome,
Crohn's disease, Dego's disease, dermatitis, dermatomyositis,
discoid lupus, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, glomerulonephritis, Grave's disease,
Guillain-Barre sydrome, Hashimoto's thyroiditis, idiopathic
pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA
nephropathy, insulin dependent diabetes (Type I), juvenile
arthritis, Meniere's disease, mixed connective tissue disease,
multiple sclerosis, myasthenia gravis, pemphigus vulgaris,
pernicious anemia, polyarteritis nodosa, polychondritis,
polyglandular syndromes, polymyalgia rheumatica, rheumatoid
arthritis, polymyositis, primary agammaglobulinemia, primary
biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's
syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's
syndrome, stiff-man syndrome, systemic lupus erythematosus,
Takayasu arteritis, temporal arteritis/giant cell arteritis,
ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's
granulomatosis.
EXAMPLES
Example 1
Identification of a Monoclonal Antibody Suitable for Capturing or
Detecting Intact C3
[0114] The following seven antihuman C3a/C3 Abs were tested in
ELISA assays as capture antibodies for intact C3 using goat
anti-C3-HRP as a detection antibody: mouse anti-C3a (Quidel, A203;
Cell Sciences HM2073, HM2074, HM2075), chicken anti-C3a(Abcam
ab48580; Genetex, GTX 78198) and goat anti-C3a (SantaCruz,
sc17237). The experiments were performed according to the protocol
described in Example 2. Considerable variability was observed in
the specificity of these antibodies. For example, as shown in FIG.
2a, the anti-C3a Ab A203 lacks specificity, it can bind C3b and
iC3b as well. Cell Science HM 2073 and HM 2075 showed good
specificity when detecting individual complement components (FIGS.
2b and 2c).
Example 2
Assay for Determining Concentration of Intact C3
[0115] This example describes a method of determining the
concentration of intact C3 in human plasma or serum samples.
General:
[0116] 1. Complement proteins, serum samples and antibody are kept
on ice; 2. During dilution, samples are mixed by slowly pipetting
up and down or inverting the tubes a few times; 3. Serum is thawed
quickly in 37.degree. C. water bath and immediately transferred to
ice; 4. Caution is exercised not to let the wells dry out during
the procedure; 5. Pipetting tips are changed during each serial
dilution.
Procedure:
[0117] 1. Plate: Thermo Scientific ultrahigh binding 96 well plate
Immulon 4HBX (Thermo Scientific, 3855); 2. Coating: Monoclonal
antibody mouse anti-C3/C3a, (Cell Sciences, HM2073) dilute stock to
2 ug/ml in PBS, add 50 ul per well, cover the plate with a plate
sealer and incubate overnight at 2-8.degree. C. or room temperature
for 1-2; 3. Blocking: Block with Pierce blocking buffer or 1% BSA
solution in PBS, add at least 200 ul to each well, incubate 1-2 h
at room temperature; 4. Standard and sample preparation: Standards
and samples will be diluted in blocking buffer. Serial dilution of
pure human C3 protein (Complement Technology, A113) is used to
generate standard curve. Wash the plate 3 times and add 50 ul of
samples to each well. Incubate 1 h at room temperature. 5.
Detection antibody: Wash plate 6 times before adding detection
antibody. Dilute Goat anti-C3-HRP (MP Biomedicals, 55237) 1:5000
with blocking buffer and add 50 ul to each well, incubate 1 h at
room temperature OR dilute chicken anti-human-C3-HRP (Immunology
Consultants Laboratory) 1: 2000 dilution add 50 ul to each well,
incubate 1 h at room temperature. 6. Substrate: Wash the plate 6
times, and add 50 ul/well of TMB substrate solution (BD Bioscience,
555214) incubate the plate for 5 to 10 min at room temperature in
the dark. Add 25 ul/well of 1M H.sub.2SO.sub.4 to stop the
reaction. Measure OD within 10 min at 450 nM.
Interpretation of Results:
Standard Curve:
[0118] A standard curve is generated for each run using healthy
human serum as diluents. The optimal dilution for each complement
component assay is determined by testing serum dilutions from
healthy subjects and patients. A dilution that yields A450 value
around 0.2-0.5 is selected. The standard curve will use the same
dilution factor to dilute healthy human serum and will use it as
diluent. The average serum absorbance is subtracted from complement
standard absorbance value to get the net absorbance value A450.
[0119] The standard curve is prepared by plotting the net
absorbance values A450 of each standard on the y-axis and
corresponding concentration indicated on the x-axis. A four
parameter curve fit (e.g., available in Microsoft Excel) is used to
generate the equation.
Calculation of Results:
[0120] The average dilution buffer absorbance is subtracted from
all the sample absorbance value to get the net absorbance value.
The four-parameter curve fitting equation is used to calculate the
unknown sample concentration (x) from the known absorbance value
(y).
Example 3
Measuring Intact C3 in Samples Containing Human Serum
[0121] The protocol described in Example 2 was used to assess C3
levels in serum samples at different dilutions to which C3 had been
added. C3 was captured using HM2073 or HM2075 anti-C3a/C3 mAb (100
ng coating per well), and detected using goat anti-C3-HRP. The data
for HM2073 are plotted in FIG. 3a. The data for HM2075 are plotted
in FIG. 3c. Tables 2-5 (FIGS. 3b and 3d) show the raw and processed
data obtained in these experiments. Rows A-H of Tables 2 and 4
correspond to wells of a 96-well plate. The samples in the wells
corresponding to row A are serially diluted 3-fold moving
downwards. Data in rows A-F, columns 1-10 is from wells containing
different concentrations of C3 as follows: row A: 1.0 .mu.g/ml; row
B 0.333 .mu.g/ml: row C: 0.111 .mu.g/ml; row D: 0.037 .mu.g/ml; row
E: 0.012 .mu.g/ml; row F: 0.004 .mu.g/ml. Data for the 0.001
.mu.g/ml concentration is not shown. Data in rows G and H is from
wells that contain matrix only (i.e., 0 .mu.g/ml C3). The average
values for the different matrices are shown in row I. BB stands for
blocking buffer.
[0122] Each set of two columns in Tables 2 and 4 represents results
from duplicate wells containing C3 at varying concentrations
ranging from 1.0 .mu.g/ml to 0 .mu.g/ml (depending on the row) in a
different matrix. The matrices are as follows: columns 1 and 2:
blocking buffer; columns 3 and 4: 0.5% serum; columns 5 and 6: 0.1%
serum; columns 7 and 8: 0.05% serum; columns 9 and 10: 0.01% serum;
columns 11 and 12: 0.5% serum. Please note that the matrices are
diluted moving downwards from row A.
[0123] The first column in Tables 3 and 5 shows the C3
concentrations corresponding to the data in each row except for the
data in the rightmost column, which is obtained from wells
containing serum only. For example, data in the top row corresponds
to wells containing C3 at 1 .mu.g/ml. Data in Tables 3 and 5 is
obtained by averaging the two corresponding values in Tables 2 and
4 and subtracting the relevant background value associated with the
matrix. For example, the value 0.0308 in the second row (labeled
0.333), fifth column (labeled C3 in 0.05% serum) of Table 3 was
obtained by averaging 0.423 and 0.424 (from row B, columns 7 and 8
of Table 2) and subtracting 0.116 (from row I, column 7 of Table
2).
Example 4
Specificity of the Assay for Intact C3
[0124] The specificity of the assay for intact C3 described in
Example 2 was tested using mock samples containing purified
complement components either individually or in mixtures.
[0125] In one set of experiments 1 .mu.g/ml each of human purified
complement proteins C3, C3b and iC3b, or 1 .mu.g/ml of C3 and 0.5
.mu.g/ml each of C3b and iC3b were loaded into the left most well
of each row of a 96-well plate. The diluent was blocking buffer.
Twofold dilutions of each sample were then made moving rightward
across the plate. The concentration of intact C3 in each sample was
measured using the assay described in Example 2. Results of a
representative assay are shown in FIG. 4a and demonstrate that
there is no significant crosstalk with either iC3b or C3b. FIG. 4b
shows results of experiments in which intact C3 was assayed in
samples that contained 1 .mu.g/ml of C3 and also contained both C3b
and iC3b protein in amounts ranging from 0.25 .mu.g/ml to 1
.mu.g/ml. FIG. 4b shows that intact C3 is accurately detected in
the presence of levels of other C3 products at relative ratios to
C3 higher than would be expected in patient samples. For example,
the readout from the sample containing 1 .mu.g/ml each of C3, iC3b,
and C3b (purple x) is almost identical to that obtained from the
sample containing 1 .mu.g/ml of C3 only (blue diamonds).
Example 5
Assay for Determining Concentration of iC3b
[0126] This example describes a method of determining the
concentration of iC3b in human plasma or serum samples.
General:
[0127] As described in Example 2.
Procedure:
[0128] 1. Assay plate: Thermo Scientific Immnulon 1 B medium
binding 96 well plate (Thermo Scientific, 3355); 2. Coating: mouse
anti-iC3b (Quidel, A209), dilute stock to 2 ug/ml in PBS, add 50 ul
per well, cover the plate with a plate sealer and incubate
overnight at 2-8.degree. C. or room temperature for 1-2; 3.
Blocking: Block with Pierce blocking buffer or 1% BSA solution in
PBS, add at least 200 ul to each well, incubate 1-2 h at room
temperature; 4. Standard and sample preparation: Standards and
samples will be diluted in blocking buffer. Serial dilution of pure
human iC3b (Complement Technology, A115) is used to generate
standard curve. Wash the plate 3 times and add 50 ul of samples to
each well. Incubate 1 h at room temperature. 5. Detection antibody:
Wash plate 6 times before adding detection antibody. Dilute Goat
anti-C3-HRP (MP Biomedicals, 55237) 1:5000 with blocking buffer and
add 50 ul to each well, incubate 1 h at room temperature. 6.
Substrate: Wash the plate 6 times, and add 50 ul/well of TMB
substrate solution (BD Bioscience, 555214) incubate the plate for 5
to 10 min at room temperature in the dark. Add 25 ul/well of 1M
H.sub.2504 to stop the reaction. Measure OD within 10 min at 450
nM.
Interpretation of Results:
[0129] As described in Example 2.
Example 6
Specificity of the iC3b Assay
[0130] The specificity of the assay for iC3b described in Example 5
was tested using mock samples containing purified complement
components either individually or in mixtures. C3 and C3b were the
C3 products found to possess the highest degree of crosstalk with
iC3b.
[0131] To demonstrate the specificity of the assay, in one set of
experiments equal amounts of human purified complement proteins C3,
C3b and iC3b were loaded into the left most well of each row of a
96-well plate. The starting concentration of each component was 10
.mu.g/ml in the leftmost well. The diluent was blocking buffer.
Twofold dilutions of each sample were then made moving rightward
across the plate. The concentration of iC3b in each sample was
measured using the assay described in Example 5. Results of a
representative assay are shown in FIG. 5 and demonstrate that there
is no significant crosstalk with either C3 or C3b when purified
components are assayed.
[0132] In another set of experiments, iC3b was assayed in samples
that contained various mixtures of C3, C3b and iC3b protein.
Results of a representative assay are shown in FIG. 6 and
demonstrate that there is no significant crosstalk with either C3
or C3b when mixtures of purified components are assayed. For
example, the sample containing 0.95 .mu.g/ml iC3b (blue diamonds)
gave virtually the same readout as the sample containing 0.95
.mu.g/ml iC3b+0.95 .mu.g/ml C3+2 .mu.g/ml C3 (purple x).
[0133] In other experiments, the assay was performed on samples
that contained iC3b and varying concentrations of serum. FIG. 7
shows a comparison of iC3b standard curves in blocking buffer to
which serum has been added. The concentration of iC3b was 2.5
.mu.g/ml in the leftmost wells of the plate (except for the 0.1%
serum condition). The data demonstrates that iC3b can be accurately
measured in samples that contain serum.
Example 7
Measuring iC3b in Human Vitreous
[0134] The assay for iC3b described in Example 5 was used to
measure iC3b in samples containing normal human vitreous to which
iC3b had been added. The vitreous was diluted to 10%, 20%, or 30%
using blocking buffer. Results are shown in FIG. 8a. The raw and
processed data are shown in Tables 6 and 7, respectively (FIG. 8b).
Please note that the data in Tables 6 and 7 is oriented differently
to that in Tables 2-5. Each set of 2 rows in Table 6 corresponds to
duplicate samples. iC3b concentration decreases moving from left to
right in columns 1-9. Columns 10-12 represent wells containing
matrix only, as indicated. Values in Table 7 are obtained by
averaging the corresponding duplicates in Table 6 and subtracting
the relevant background value for matrix. For example, the value
0.049 in the row labeled `iC3b in 20% vitreous` and column labeled
`0.0125` iC3b concentration in Table 7 is obtained by averaging
0.361 and 0.384 from column 5, rows E and F of Table 6, and
subtracting 0.328 (the `matrix only` value for 20% vitreous in
Table 6).
Example 8
Assay for Determining Concentration of Total C3
[0135] This example describes a method of determining the
concentration of total C3 in human plasma or serum samples.
DEFINITIONS/ABBREVIATIONS
[0136] PBS Phosphate Buffered Saline
[0137] TMB Tetramethylbenzidine
Materials and Instruments
[0138] 1. Immulon 1B flat bottom Medium Binding 96-well plates
(Thermo Electron Corporation, Cat #3355) [0139] 2. Anti-human C3
HRP-conjugated polyclonal antibody (Cappel 55237) [0140] 3. Murine
anti-human C3d monoclonal antibody (Quidel, Cat# A207) [0141] 4.
Goat anti-mouse IgG HRP antibody (Jackson Immuno Labs, Cat
#115-035-003) [0142] 5. Blocking buffer: Starting block (PBS)
Blocking Buffer (Thermo Electron Corporation, Cat#37538) [0143] 6.
TMB Substrate Reagent Set (BD Bioscience, Cat#555214) [0144] 7.
1.times.PBS [0145] 8. Wash buffer: 1.times.PBS with 0.05% Tween-20
[0146] 9. 1M H.sub.2SO.sub.4 [0147] 10. Plate reader capable of
measuring absorbance at 450 nm
Procedure:
[0148] Assay Preparation (for One 96-Well Plate) [0149] Capture
Antibody: Dilute murine anti-human C3d antibody in 5 mL PBS for a
final solution concentration of 2 ug/ml. [0150] Gently mix antibody
by pipetting [0151] Coat an Immulon medium binding plate with 50
.mu.l/well capture antibody in columns 1-12 and in duplicate (2)
rows for each sample to be tested. Seal the plate and incubate
overnight at 4.degree. C. or at room temperature for 1 hour. [0152]
Shake and tap the plate to remove the coating antibody. [0153]
Block the plate with 200 .mu.l per well of blocking buffer for 1
hour at room temperature. [0154] Shake off the blocking buffer and
wash the plate by filling with at least 300 .mu.l/well washing
buffer and then decant. Wash it 6.times. manually or by automatic
plate washer.
[0155] Assay Procedure [0156] Dilute human C3 protein in blocking
buffer with a 5 .mu.g/mL final concentration for the working
dilution. [0157] Dilute human serum or plasma in blocking buffer in
a 1:250 dilution for the working dilution. [0158] Add 200 .mu.l of
working sample or standard into the first well (column 1) of each
row. From well 2 to 12, add 100 .mu.l of blocking buffer. Serially
dilute the samples by removing 100 ul of each standard or sample
from column 1 of each row and adding it to the next well (column
2). Mix by pipetting 10 times. Continue this procedure for each
subsequent well, stopping with column 11. Column 12 should remain
as the blank, containing only blocking buffer. Cover the plate with
plate sealer and incubate for 1 hour at room temperature. [0159]
Shake off samples and wash 6.times. as described above [0160]
Antibody Detection solution: Dilute 1 .mu.l Anti-human C3
HRP-conjugated polyclonal antibody in 5 mL blocking buffer for a
1:5000 final concentration. [0161] Add 50 .mu.l/well of anti-C3
antibody detection solution, and incubate for 1 hour at room
temperature. [0162] Shake off antibody and wash 6.times. as
described above. [0163] Detection of reaction using TMB
substrate--mix 5 mL of reagent A with 5 mL reagent B per test
plate, no more than 10 minutes prior to use. Avoid exposure to
light. [0164] Add 100 .mu.l/well of TMB substrate. Incubate for 2-5
minutes or until darkest color is approximately 1.2 OD. [0165] Stop
the reaction by adding 50 .mu.l/well of 1M H2SO.sub.4 [0166] Read
absorbance on plate reader at 450 nm wavelength.
Example 9
Assessing Intact C3 Levels in Individuals with AMD versus Controls
Protocol
DEFINITIONS/ABBREVIATIONS/CHEMICALS
[0167] PBS: Phosphate buffered saline without Ca++ and Mg++ EDTA:
Ethylenediaminetetraacetic acid TMB: Tetramethylenbenzidine
substrate solution
H2SO4: Sulfuric Acid
[0168] Materials and Instruments
10.times.PBS: 10.times. Phosphate buffered saline without Ca++ and
Mg++. (Cellgro, Mediatech Inc, Cat #: 46-013-CM). Plate sealer:
Denville Scientific, Inc, # B1212-45
EDTA: Gibco Invitrogen Corporation #15575-038
Tween 20: Molecular Biology Grade, (Promega Corporation Cat #
H5151).
Blocking Buffer: Thermo Scientific #37538
[0169] ELISA plate: Thermo Electron Corporation #3855 Mouse
anti-human C3a/C3 Cell Sciences, #HM2075
Human C3: Complement Technologies, Inc #A113
[0170] Goat anti human C3-HRP: MP Biomedicals #55237
TMB: BD OptEIA, BD Biosciences Cat#555214)
H2SO4: 1M Sulfuric Acid
[0171] BIO-TEK ELX-405 ELISA microplate washer BMG microplate
reader
[0172] Procedure
[0173] 0. General
0.1 Complement protein, serum samples and antibodies are kept on
ice. 0.2 Avoid vortexing the samples during dilution, instead mix
by slowly pipetting up and down or inverting the tubes a few times.
0.3 Thaw serum quickly in 37.degree. C. water bath, and immediately
transfer it to ice. 0.4 Do not let the wells dry out during the
procedure. 0.5 Change pipetter tips during each serial
dilution.
[0174] 1.1 Prepare the reagents and cocktails as detailed below
(for one ELISA plate):
1.1.1 Preparation of stock 1.times.PBS Dilute 100 ml 10.times.PBS
stock in 900 ml distilled water 1.1.2 Preparation of stock Wash
Buffer Add 100 mL of 10.times.PBS and 0.5 mL Tween-20 to each 900
mL distilled water 1.1.3 Preparation of stock PBS-Tw-EDTA Add 0.5
ml of Tween-20 and 20 mL of 500 mM EDTA stock to each 980 mL
1.times.PBS. 1.1.4 Preparation of coating antibody solution Dilute
64 of mouse anti-human C3a/C3 (stock 0.1 mg/mL) into 6 mL of
1.times.PBS. 1.1.5 Preparation of C3 Standard (4 ug/mL) Dilute 4
.mu.l of human C3 (stock 1.0 mg/mL) into 1 mL of PBS-Tw-EDTA. 1.1.6
Preparation of detection antibody solution Dilute 3 .mu.l of goat
anti C3-HRP into 6 mL of blocking buffer. 1.1.7 Preparation of TMB
substrate Prepare immediately before use, mix 3 mL of Reagent A
with 3 mL of Reagent B.
[0175] 1.2 Plate Coating:
1.2.1 Add 60 .mu.l of the coating antibody solution to all wells,
cover and incubate at 4.degree. C. overnight or room temperature
for 2 hours. 1.2.2 Throw out coating and tap on paper towels to
dry.
[0176] 1.3 Blocking
1.3.1 Add 200 .mu.L of blocking buffer per well 1.3.2 Cover and
incubate for 1-2 hours at room temperature 1.3.3 Wash plate 3 times
with wash buffer
[0177] 1.4 Preparing for Calibration Curve
1.4.1 C3 standard curve will be generated in triplicate or
quadruplicate. All samples and standards will be diluted in
PBS-Tw-EDTA. All serial dilutions will be done in tubes and then
transferred to the plate. In seven empty 1.7 mL tubes add 300 ul of
PBS-Tw-EDTA to each. Then add 300 ul of the standard stock solution
to the first tube and mix five times. Change tips and take 300 ul
from the first tube and add it to the second tube, mix five times
and change tips. Continue until serial dilutions are complete. Add
50 ul of the dilutions to the wells as described in the template
below:
TABLE-US-00002 1 2 3 4 5 6 7 8 9 10 11 12 A SD1 SD1 SD1 B SD2 SD2
SD2 C SD3 SD3 SD3 D SD4 SD4 SD4 E SD5 SD5 SD5 F SD6 SD6 SD6 G SD7
SD7 SD7 H BC BC BC SD: Standard Dilution BC: Blank Control
1.4.2 Cover and incubate at room temperature for 1 hour. 1.4.3 Wash
plate 6 times with wash buffer
[0178] 1.5 Add HRP Conjugated Detection Antibody
1.5.1 Add 50 .mu.l per well of the detection antibody solution and
incubate plate at room temperature for 1 hour. 1.5.2 Wash plate 6
times with wash buffer.
[0179] 1.6 Add Substrate
[0180] Add 50 .mu.l of TMB substrate to each well and incubate for
colorimetric reaction (5 to 15 minutes depending on sample
concentration) Add 25 .mu.l of 1M H2SO4 to each well to stop the
reaction, and measure the absorbance at 450 nm;
[0181] 1.7 Data Analysis
1.7.1 Absorbance versus concentration for the standard curve will
be fitted to a four-parameter logistic regression (omit the first
dilution of 2 .mu.g/mL). Use the equation to back fit results to
find the concentration of C3 in samples.
[0182] Results
[0183] The protocol set forth immediately above was used to assess
intact C3 levels in serum samples obtained from individuals who had
been diagnosed AMD (and in some cases their medications included
Lucentis and/or anti-AMD vitamin preparations) and in control serum
samples obtained from individuals more than 60 years of age who had
not been diagnosed with AMD (but in at least some cases suffered
from one or more other diseases). The samples were purchased from
Bioreclamation, Inc. (Liverpool, N.Y.). The two sets of
measurements were compared. FIG. 9 shows results of the assay. The
values for three AMD and two non-AMD samples were out of range and
were not included in the analysis.
[0184] It was found that levels of intact C3 were significantly
higher in samples from individuals with AMD than in control
samples. Levels of intact C3 in samples from individuals with AMD
ranged between 314.4 .mu.g/ml and 570.1 .mu.g/m, while levels of
intact C3 in samples from control individuals ranged between 10.18
.mu.g/ml and 173.9 .mu.g/ml. The intact C3 concentrations for the
individual samples in .mu.g/ml are shown below. The data represent
the average of several replicates. Average values and standard
deviations are readily calculated.
TABLE-US-00003 TABLE AMD Control Samples Samples 457.1 146.5 n/a
n/a 348.2 5.734 417 108.6 383.5 72.16 282.8 13.83 374.6 74.15 369.4
13.27 314.3 173.9 325.9 20.39 540.9 14.81 n/a 18.95 n/a 14.79 570.2
6.116 545.6 10.18 430.1 7.504 479.5 9.834 506.2 125.2 420.2
10.9
[0185] The assay was repeated using the same set of samples about 2
months later. The samples were divided into two sets of 10 AMD
samples and two sets of 10 control samples. As shown in FIGS. 10a
and 10b, similar results were obtained except that for one control
sample, the intact C3 concentration was within the range measured
for AMD samples. The intact C3 concentrations for the individual
samples in .mu.g/ml are shown below. The data represent the average
of several replicates. Average values and standard deviations are
readily calculated.
TABLE-US-00004 1 through 10 AMD Control Samples Samples 1 2 239.356
72.002 255.739 214.855 269.789 14.89 233.476 65.376 284.729 102.903
196.58 100.267 234.449 59.131 224.373 14.2 268.204 16.151 225.902
104.644 11 through 20 AMD Control 1 2 323.715 12.888 374.919 21.316
488.163 18.443 311.757 10.245 290.958 19.119 372.779 8.7786 365.226
17.354 343.141 73.758 271.581 12.94 287.322 11.764
[0186] The above results show that individuals with AMD can be
distinguished from those not having AMD based on serum
concentrations measured using the assay.
Example 10
Assay for Complement Activation in Individuals with Early AMD
[0187] Serum samples are obtained from a group of subjects with
early AMD. The methods described in Examples 2 and 4 are used to
measure intact C3 and iC3b in each of the samples. For each sample,
the ratio of iC3b to intact C3 is obtained to assess the extent of
complement activation. The subjects are monitored over the
subsequent 3 years for progression, e.g., to advanced AMD
(geographic atrophy and/or exudative AMD). During this time period,
serum samples are obtained at intervals and complement activation
is assessed by determining the ratio of iC3b to intact C3.
Correlations are made between the extent of complement activation
and the rapidity and/or likelihood of progression to advanced AMD.
The information is used to develop a test to determine the
likelihood of progression from early to advanced AMD.
Example 11
Assay for Intact C3 Levels in Individuals with AMD
[0188] Serum samples are obtained from between 50 and 5000 subjects
exhibiting various stages of AMD (AREDS 2-4) and from between 50
and 5000 subjects at least 55 years of age who do not have AMD
(AREDS 1). The subjects may be screened, and those will illnesses
or other conditions that may acutely affect the level of intact C3
or the accuracy of the assay may be excluded. The method described
in Example 10 is used to measure the level of intact C3 in the
samples. Correlations between the level of intact C3 and AMD stage
are determined. Standard statistical methods may be used. The
subjects without AMD are monitored over the subsequent 3 years for
development of clinically evident AMD. The subjects with AMD are
monitored over the subsequent 3 years for progression, e.g., from
AREDS 2 stage to advanced AMD (with geographic atrophy and/or
exudative AMD). During this time period, serum samples are obtained
at intervals (e.g., every 1-3 months) and the level of intact C3 is
assessed. Correlations between the level of intact C3 and the
rapidity and/or likelihood of progression to advanced AMD are
determined. Standard statistical methods may be used. Analyses are
performed on the entire subject groups and on subsets composed of
subjects with particular risk factors such as smoking or genetic
background or in different age brackets. For example, analyses are
performed on subjects with genotypes associated with AMD risk
(e.g., having various CFH polymorphisms). The information is used
to further refine the methods of using the level of intact C3 to
provide improved prognostic, diagnostic, and/or therapeutic
information relating to AMD development or progression.
[0189] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the embodiments described above, but rather is as set
forth in the claims. In the claims articles such as "a,", "an" and
"the" may mean one or more than one unless indicated to the
contrary or otherwise evident from the context. Claims or
descriptions that include "or" between one or more members of a
group are considered satisfied if one, more than one, or all of the
group members are present in, employed in, or otherwise relevant to
a given product or process unless indicated to the contrary or
otherwise evident from the context. The invention provides
embodiments in which exactly one member of the group is present in,
employed in, or otherwise relevant to a given product or process.
The invention also provides embodiments in which more than one, or
all of the group members are present in, employed in, or otherwise
relevant to a given product or process. Furthermore, it is to be
understood that the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, descriptive terms, etc., from one or more of the
listed claims is introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more elements or limitations found in any other claim that
is dependent on the same base claim. Furthermore, where the claims
recite a composition, it is to be understood that methods of
administering the composition according to any of the methods
disclosed herein, and methods of using the composition for any of
the purposes disclosed herein are provided, and methods of making
the composition according to any of the methods of making disclosed
herein are provided, unless otherwise indicated or unless it would
be evident to one of ordinary skill in the art that a contradiction
or inconsistency would arise. It should also be understood that
references herein to "the invention" or to materials of use in the
invention or to applications or uses of the invention can refer to
one embodiment or aspect of the invention, or to more than one
embodiment or aspect of the invention, and should not be
interpreted as limiting.
[0190] Where elements are presented as lists or groups, it is to be
understood that each subgroup of the elements is also disclosed,
and any element(s) can be removed from the group. For purposes of
conciseness only some of these embodiments have been specifically
recited individually and specifically herein, but the invention
includes all such embodiments. It should also be understood that,
in general, where the invention, or aspects of the invention,
is/are referred to as comprising particular elements, features,
etc., certain embodiments of the invention or aspects of the
invention consist, or consist essentially of, such elements,
features, etc.
[0191] In certain embodiments of any of the methods of the
invention for assessing complement activation or proteolytic
activity, the method further can comprise a step of providing a
sample obtained from subject suffering from or at risk of a
complement deficiency or a complement-mediated disorder or clinical
condition. In certain embodiments of any of the methods of the
invention for assessing complement activation or proteolytic
activity, the method further can comprise diagnosing a subject as
suffering from or at risk of a complement deficiency or a
complement-mediated disorder or clinical condition. In certain
embodiments of any of the methods of the invention for assessing
complement activation or proteolytic activity, the method can
further comprise administering a complement modulating agent to the
subject.
[0192] Where ranges are given herein, the invention provides
embodiments in which the endpoints are included, embodiments in
which both endpoints are excluded, and embodiments in which one
endpoint is included and the other is excluded. It should be
assumed that both endpoints are included unless indicated
otherwise. Furthermore, it is to be understood that unless
otherwise indicated or otherwise evident from the context and
understanding of one of ordinary skill in the art, values that are
expressed as ranges can assume any specific value or subrange
within the stated ranges in different embodiments of the invention,
to the tenth of the unit of the lower limit of the range, unless
the context clearly dictates otherwise. A time period of 1 month is
understood to mean 30 days. A time period of 1 year is understood
to mean 365 days. For any embodiment of the invention in which a
numerical value is prefaced by "about" or "approximately", the
invention provides an embodiment in which the exact value is
recited. For any embodiment of the invention in which a numerical
value is not prefaced by "about" or "approximately", the invention
provides an embodiment in which the value is prefaced by "about" or
"approximately".
[0193] Any particular embodiment, feature, or aspect of the present
invention may be explicitly excluded from any one or more of the
claims.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 36 <210> SEQ ID NO 1 <400> SEQUENCE: 1 000
<210> SEQ ID NO 2 <400> SEQUENCE: 2 000 <210> SEQ
ID NO 3 <400> SEQUENCE: 3 000 <210> SEQ ID NO 4
<400> SEQUENCE: 4 000 <210> SEQ ID NO 5 <400>
SEQUENCE: 5 000 <210> SEQ ID NO 6 <400> SEQUENCE: 6 000
<210> SEQ ID NO 7 <400> SEQUENCE: 7 000 <210> SEQ
ID NO 8 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
N-terminal H <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (13)..(13) <223> OTHER INFORMATION:
C-terminal CONH2 <400> SEQUENCE: 8 Ile Cys Val Val Gln Asp
Trp Gly His His Arg Cys Thr 1 5 10 <210> SEQ ID NO 9
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: N-terminal
ACETYLATION <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (13)..(13) <223> OTHER INFORMATION:
C-terminal CONH2 <400> SEQUENCE: 9 Ile Cys Val Val Gln Asp
Trp Gly His His Arg Cys Thr 1 5 10 <210> SEQ ID NO 10
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: N-terminal
ACETYLATION <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (13)..(13) <223> OTHER INFORMATION:
C-terminal CONH2 <400> SEQUENCE: 10 Ile Cys Val Tyr Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 <210> SEQ ID NO 11
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: N-terminal
ACETYLATION <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (13)..(13) <223> OTHER INFORMATION:
C-terminal COOH <400> SEQUENCE: 11 Ile Cys Val Trp Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 <210> SEQ ID NO 12
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: N-terminal
ACETYLATION <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (13)..(13) <223> OTHER INFORMATION:
C-terminal CONH2 <400> SEQUENCE: 12 Ile Cys Val Trp Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 <210> SEQ ID NO 13
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: N-terminal
ACETYLATION <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (13)..(13) <223> OTHER INFORMATION:
d-Thr <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (13)..(13) <223> OTHER INFORMATION:
C-terminal COOH <400> SEQUENCE: 13 Ile Cys Val Trp Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 <210> SEQ ID NO 14
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: N-terminal
ACETYLATION <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (4)..(4) <223> OTHER INFORMATION:
2-napthylalanine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (13)..(13) <223> OTHER INFORMATION:
C-terminal CONH2 <400> SEQUENCE: 14 Ile Cys Val Ala Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 <210> SEQ ID NO 15
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: N-terminal
ACETYLATION <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (4)..(4) <223> OTHER INFORMATION:
2-naphthylalanine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: C-terminal COOH <400> SEQUENCE: 15 Ile Cys Val
Ala Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10 <210> SEQ ID
NO 16 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
N-terminal ACETYLATION <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: 1-naphthylalanine <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (13)..(13) <223>
OTHER INFORMATION: C-terminal COOH <400> SEQUENCE: 16 Ile Cys
Val Ala Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10 <210> SEQ
ID NO 17 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
N-terminal ACETYLATION <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: 2-indanylglycine carboxylic acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (13)..(13)
<223> OTHER INFORMATION: C-terminal CONH2 <400>
SEQUENCE: 17 Ile Cys Val Gly Gln Asp Trp Gly Ala His Arg Cys Thr 1
5 10 <210> SEQ ID NO 18 <211> LENGTH: 13 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal ACETYLATION <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: 2-indanylglycine carboxylic acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 18 Ile Cys Val Gly Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 19 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATOIN <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: dihydrotrpytophan
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 19 Ile Cys Val Trp Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 20 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 4-benzoyl-L-phenylalanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 20 Ile Cys Val Phe Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 21 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: p-benzoyl-L-phenylalanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 21 Ile Cys Val Phe Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 22 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Bta <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (13)..(13)
<223> OTHER INFORMATION: C-terminal COOH <400>
SEQUENCE: 22 Ile Cys Val Xaa Gln Asp Trp Gly Ala His Arg Cys Thr 1
5 10 <210> SEQ ID NO 23 <211> LENGTH: 13 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal ACETYLATION <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: Bta <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (13)..(13) <223>
OTHER INFORMATION: C-terminal CONH2 <400> SEQUENCE: 23 Ile
Cys Val Xaa Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10 <210>
SEQ ID NO 24 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal ACETYLATION <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (9)..(9)
<223> OTHER INFORMATION: 2-alpha-aminobutyric acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 24 Ile Cys Val Trp Gln Asp Trp Gly Xaa
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 25 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal H <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: C-terminal COOH <400>
SEQUENCE: 25 Gly Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys
Thr Ala Asn 1 5 10 15 <210> SEQ ID NO 26 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 5-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 26 Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 27 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 5-methyl-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 27 Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 28 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal Ac <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: 1-methyl-L-tryptophan <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal CONH2
<400> SEQUENCE: 28 Ile Cys Val Trp Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 29 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(7)..(7) <223> OTHER INFORMATION: 5-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 29 Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 30 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 5-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (7)..(7) <223> OTHER INFORMATION:
5-fluoro-L-tryptophan <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: C-terminal CONH2 <400> SEQUENCE: 30 Ile Cys Val
Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10 <210> SEQ ID
NO 31 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
N-terminal ACETYLATION <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: 5-methyl-L-tryptophan <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (7)..(7) <223> OTHER
INFORMATION: 5-fluoro-L-tryptophan <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (13)..(13) <223>
OTHER INFORMATION: C-terminal CONH2 <400> SEQUENCE: 31 Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10 <210>
SEQ ID NO 32 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal ACETYLATION <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: 1-methyl-L-tryptophan <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(7)..(7) <223> OTHER INFORMATION: 5-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 32 Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 33 <211> LENGTH:
15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-term H <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: 6-fluoro-L-tryptophan <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(8)..(8) <223> OTHER INFORMATION: 6-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (15)..(15) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 33 Gly Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Asn 1 5 10 15 <210> SEQ ID NO 34 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: N-terminal ACETYLATION
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: 1-formyl-Trp
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 34 Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 35 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal acetylation <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 1-methoxy-tryptophan
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 35 Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 36 <211> LENGTH:
15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-term H <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: 5-fluoro-L-tryptophan <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(8)..(8) <223> OTHER INFORMATION: 5-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (15)..(15) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 36 Gly Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Asn 1 5 10 15
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 36 <210>
SEQ ID NO 1 <400> SEQUENCE: 1 000 <210> SEQ ID NO 2
<400> SEQUENCE: 2 000 <210> SEQ ID NO 3 <400>
SEQUENCE: 3 000 <210> SEQ ID NO 4 <400> SEQUENCE: 4 000
<210> SEQ ID NO 5 <400> SEQUENCE: 5 000 <210> SEQ
ID NO 6 <400> SEQUENCE: 6 000 <210> SEQ ID NO 7
<400> SEQUENCE: 7 000 <210> SEQ ID NO 8 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: N-terminal H <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal CONH2
<400> SEQUENCE: 8 Ile Cys Val Val Gln Asp Trp Gly His His Arg
Cys Thr 1 5 10 <210> SEQ ID NO 9 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal CONH2
<400> SEQUENCE: 9 Ile Cys Val Val Gln Asp Trp Gly His His Arg
Cys Thr 1 5 10 <210> SEQ ID NO 10 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal CONH2
<400> SEQUENCE: 10 Ile Cys Val Tyr Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 11 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 11 Ile Cys Val Trp Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 12 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal CONH2
<400> SEQUENCE: 12 Ile Cys Val Trp Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 13 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: d-Thr <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 13 Ile Cys Val Trp Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 14 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 2-napthylalanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 14 Ile Cys Val Ala Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 15 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 2-naphthylalanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 15 Ile Cys Val Ala Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10
<210> SEQ ID NO 16 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal ACETYLATION <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: 1-naphthylalanine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 16 Ile Cys Val Ala Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 17 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 2-indanylglycine carboxylic
acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 17 Ile Cys Val Gly Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 18 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 2-indanylglycine carboxylic
acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 18 Ile Cys Val Gly Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 19 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATOIN <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: dihydrotrpytophan
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 19 Ile Cys Val Trp Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 20 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 4-benzoyl-L-phenylalanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 20 Ile Cys Val Phe Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 21 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: p-benzoyl-L-phenylalanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 21 Ile Cys Val Phe Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 22 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Bta <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (13)..(13)
<223> OTHER INFORMATION: C-terminal COOH <400>
SEQUENCE: 22 Ile Cys Val Xaa Gln Asp Trp Gly Ala His Arg Cys Thr 1
5 10 <210> SEQ ID NO 23 <211> LENGTH: 13 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal ACETYLATION <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: Bta <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (13)..(13) <223>
OTHER INFORMATION: C-terminal CONH2 <400> SEQUENCE: 23 Ile
Cys Val Xaa Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10 <210>
SEQ ID NO 24 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal ACETYLATION <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (9)..(9)
<223> OTHER INFORMATION: 2-alpha-aminobutyric acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 24 Ile Cys Val Trp Gln Asp Trp Gly Xaa
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 25 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic
peptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
N-terminal H <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (16)..(16) <223> OTHER INFORMATION:
C-terminal COOH <400> SEQUENCE: 25 Gly Ile Cys Val Trp Gln
Asp Trp Gly Ala His Arg Cys Thr Ala Asn 1 5 10 15 <210> SEQ
ID NO 26 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
N-terminal ACETYLATION <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: 5-fluoro-L-tryptophan <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (13)..(13) <223>
OTHER INFORMATION: C-terminal CONH2 <400> SEQUENCE: 26 Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10 <210>
SEQ ID NO 27 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal ACETYLATION <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: 5-methyl-L-tryptophan <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal CONH2
<400> SEQUENCE: 27 Ile Cys Val Trp Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 28 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal Ac <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: 1-methyl-L-tryptophan <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal CONH2
<400> SEQUENCE: 28 Ile Cys Val Trp Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 29 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(7)..(7) <223> OTHER INFORMATION: 5-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 29 Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 30 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-terminal ACETYLATION <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: 5-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (7)..(7) <223> OTHER INFORMATION:
5-fluoro-L-tryptophan <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: C-terminal CONH2 <400> SEQUENCE: 30 Ile Cys Val
Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10 <210> SEQ ID
NO 31 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
N-terminal ACETYLATION <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: 5-methyl-L-tryptophan <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (7)..(7) <223> OTHER
INFORMATION: 5-fluoro-L-tryptophan <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (13)..(13) <223>
OTHER INFORMATION: C-terminal CONH2 <400> SEQUENCE: 31 Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10 <210>
SEQ ID NO 32 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal ACETYLATION <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: 1-methyl-L-tryptophan <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(7)..(7) <223> OTHER INFORMATION: 5-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION: C-terminal
CONH2 <400> SEQUENCE: 32 Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 <210> SEQ ID NO 33 <211> LENGTH:
15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-term H <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: 6-fluoro-L-tryptophan <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(8)..(8) <223> OTHER INFORMATION: 6-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (15)..(15) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 33 Gly Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Asn 1 5 10 15 <210> SEQ ID NO 34 <211>
LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal ACETYLATION <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: 1-formyl-Trp <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (13)..(13)
<223> OTHER INFORMATION: C-terminal CONH2 <400>
SEQUENCE: 34 Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1
5 10 <210> SEQ ID NO 35 <211> LENGTH: 13 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: N-terminal acetylation <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: 1-methoxy-tryptophan <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(13)..(13) <223> OTHER INFORMATION: C-terminal CONH2
<400> SEQUENCE: 35 Ile Cys Val Trp Gln Asp Trp Gly Ala His
Arg Cys Thr 1 5 10 <210> SEQ ID NO 36 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: N-term H <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: 5-fluoro-L-tryptophan <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(8)..(8) <223> OTHER INFORMATION: 5-fluoro-L-tryptophan
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (15)..(15) <223> OTHER INFORMATION: C-terminal COOH
<400> SEQUENCE: 36 Gly Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Asn 1 5 10 15
* * * * *
References