U.S. patent application number 14/847761 was filed with the patent office on 2016-03-10 for methods for conducting multiplexed assays.
This patent application is currently assigned to MESO SCALE TECHNOLOGIES, LLC. The applicant listed for this patent is MESO SCALE TECHNOLOGIES, LLC. Invention is credited to Eli N. Glezer, Sudeep Kumar, Pankaj Oberoi, George Sigal, Michael Tsionsky.
Application Number | 20160069872 14/847761 |
Document ID | / |
Family ID | 51488514 |
Filed Date | 2016-03-10 |
United States Patent
Application |
20160069872 |
Kind Code |
A1 |
Glezer; Eli N. ; et
al. |
March 10, 2016 |
METHODS FOR CONDUCTING MULTIPLEXED ASSAYS
Abstract
The invention relates to methods for conducting solid-phase
binding assays. One example is an assay method having improved
analyte specificity where specificity is limited by the presence of
non-specific binding interactions.
Inventors: |
Glezer; Eli N.; (Del Mar,
CA) ; Kumar; Sudeep; (Gaithersburg, MD) ;
Oberoi; Pankaj; (Rockville, MD) ; Sigal; George;
(Rockville, MD) ; Tsionsky; Michael; (Derwood,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MESO SCALE TECHNOLOGIES, LLC |
Rockville |
MD |
US |
|
|
Assignee: |
MESO SCALE TECHNOLOGIES,
LLC
Rockville
MD
|
Family ID: |
51488514 |
Appl. No.: |
14/847761 |
Filed: |
September 8, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62047097 |
Sep 8, 2014 |
|
|
|
Current U.S.
Class: |
506/9 ; 506/16;
536/23.1 |
Current CPC
Class: |
B01L 2300/043 20130101;
G01N 33/6845 20130101; B01L 3/50853 20130101; G01N 33/54306
20130101; C12Q 1/6804 20130101; B01L 2300/021 20130101; B01L
2300/0829 20130101; B01L 2300/0609 20130101; G01N 2458/10 20130101;
G01N 33/54353 20130101; C12Q 1/6816 20130101; C12Q 1/6876 20130101;
C12Q 1/6816 20130101; C12Q 2563/131 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/68 20060101 G01N033/68; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of conducting a multiplexed binding assay for a
plurality of analytes of interest comprising (a) combining, in one
or more steps, the following components: (i) a sample comprising a
first analyte of interest and a second analyte of interest, (ii) a
first targeting agent immobilized on a first binding domain, (iii)
a first targeting agent complement connected to a linking agent,
wherein the first targeting agent complement is a binding partner
of the first targeting agent, (iv) a first binding reagent
connected to a supplemental linking agent, wherein the first
binding reagent is a binding partner of the first analyte, (v) a
second targeting agent immobilized on a second binding domain, (vi)
a second targeting agent complement connected to a linking agent,
wherein the second targeting agent complement is a binding partner
of the second targeting agent, (vii) a second binding reagent
connected to a supplemental linking agent, wherein the second
binding reagent is a binding partner of the second analyte, and
(viii) optionally, at least two copies of a bridging agent,
wherein, if the bridging agent is omitted, each linking agent is a
binding partner of the supplemental linking agent, or if the
bridging agent is included, the bridging agent has a first binding
site for one of the linking agents and an additional binding site
for one of the supplemental linking agents; (b) forming (ii) a
first binding complex on the first binding domain comprising the
first targeting agent, the first targeting agent complement, the
first binding reagent and the first analyte, and (iii) a second
binding complex on the second binding domain comprising the second
targeting agent, the second targeting agent complement, the second
binding reagent and the second analyte, and (c) measuring the
amount of the first and second analytes on the first and second
binding domains, respectively.
2. The method of claim 1, wherein the sample contains one or more
additional analytes of interest and for each additional analyte of
interest, the combining step (a) further comprises combining, in
one or more steps, (ix) an additional targeting agent immobilized
on an additional binding domain, an additional targeting agent
complement connected to a linking agent, and an additional binding
reagent connected to a supplemental linking agent, and (x) an
additional binding complex on the additional binding domain
comprising the additional targeting agent, the additional targeting
agent complement, the additional binding reagent and the additional
analyte; the forming step (b) further comprises forming (iii) an
additional binding complex on the additional binding domain
comprising the additional targeting agent, the additional targeting
agent complement, the additional binding reagent and the additional
analyte; and the measurement in step (c) further comprises
measuring the amount of the additional analyte on the additional
binding domain.
3. The method of claim 1, wherein the first targeting agent
complement and the first binding reagent are provided as a
pre-bound first targeting complex comprising the first targeting
agent complement and the first binding reagent linked through a
binding interaction between the linking agent and supplemental
linking agent; and the second targeting agent complement and the
second binding reagent are provided as a pre-bound second targeting
complex comprising the second targeting agent complement and the
second binding reagent linked through a binding interaction between
the linking agent and supplemental linking agent.
4. The method of claim 3, wherein the first targeting complex is
provided pre-bound to the first targeting agent immobilized on the
first binding domain; and the second targeting complex is provided
pre-bound to the second targeting agent immobilized on the second
binding domain.
5. The method of claim 1, wherein step (a) includes the steps of:
combining, in a first volume of liquid, said first targeting agent
complement, said first binding reagent and, if used, said bridging
reagent and linking said first targeting agent complement and said
first binding reagent through their attached linking agents to form
a first targeting complex; and combining, in a second volume of
liquid, said second targeting agent complement, said second binding
reagent and, if used, said bridging reagent and linking said second
targeting agent complex complement and said second binding reagent
through their attached linking agents to form a second targeting
complex.
6. The method of claim 5, wherein step (a) further includes the
steps of: combining said first and second targeting complexes,
contacting the combination of said first and second targeting
complexes with said first and second binding domains, and binding
said first targeting complex to said first targeting agent on said
first binding domain and binding said second targeting complex to
said second targeting agent on said second binding domain.
7. The method of claim 5, wherein combining step (a) further
includes: combining the first and second targeting complexes with
the sample to form a mixture thereof, binding the first analyte to
the first binding reagent in the first targeting complex and
binding the second analyte to the second binding reagent in the
second targeting complex, contacting a mixture of the first and
second targeting complexes bound to first and second analytes,
respectively, with the first and second binding domains; and
binding the first targeting complex to the first targeting agent on
the first binding domain and binding the second targeting complex
to the second targeting agent on the second binding domain.
8. The method of claim 7, wherein the first and second targeting
complexes are combined with the sample prior to contacting the
first and second targeting complexes with the first and second
binding domains.
9. The method of claim 7, wherein the first and second targeting
complexes are combined with the sample after contacting the first
and second targeting complexes with the first and second binding
domains.
10. The method of claim 7, wherein the first and second targeting
complexes are combined with the sample and contacted with the first
and second binding domains at the same time.
11. The method of claim 4, wherein step (a) further includes the
steps of: contacting said first and second targeting complexes on
said first and second binding domains with said sample, binding
said first analyte to the first binding reagent in said first
targeting complex and binding said second analyte to said second
binding reagent in said second targeting complex.
12. The method of claim 1, wherein the bridging agent is omitted
and the linking agent is bound to the supplemental linking agent
through a binding interaction between (a) a thiol group and a
maleimide or iodoacetamide groups; (b) an aldehyde and a hydrazide;
or (c) an alkyne and an azide.
13. The method of claim 1, wherein the bridging agent is omitted
and (a) the linking agent is biotin and supplemental linking agent
is streptavidin or avidin; (b) the linking agent is streptavidin or
avidin and the supplemental linking agent is biotin; (c) the
linking agent is a peptide and the supplemental linking agent is an
anti-peptide antibody; or (d) the linking agent is an anti-peptide
antibody and the supplemental linking agent is a peptide.
14. The method of claim 1, wherein the bridging agent is included,
the bridging agent is streptavidin or avidin, and the linking
agents and the supplemental linking agents are each biotin.
15. The method of claim 1, wherein the first and second binding
reagents each comprise a receptor, ligand, antibody, hapten,
antigen, epitope, mimitope, aptamer, or an intercalater capable of
binding to said first and second analytes, respectively.
16. The method of claim 1, wherein the first and second binding
reagents are antigens.
17. The method of claim 1, wherein the first and second binding
reagents are antibodies.
18. The method of claim 1, wherein the method comprises conducting
a sandwich binding assay.
19. The method of claim 1, wherein the components combined in step
(a) further comprise a first detection reagent that binds the first
analyte and a second detection reagent that binds the second
analyte, and the first and second complexes formed in step (b)
further comprise the first and second detection reagents,
respectively.
20. The method of claim 19, wherein the first and second detection
reagents each comprise a detectable label.
21. The method of claim 19, wherein the first and second binding
reagents and the first and second detection reagents are
antibodies.
22. The method of claim 19, wherein the first and second detection
reagents can bind to either the first or second analyte.
23. The method of claim 22, wherein the first and second detection
reagents each comprise a detectable label.
24. The method of claim 22, wherein the first and second binding
reagents are antigens, the analytes are antibodies against the
antigens and the detection reagents comprise anti-immunoglobulin
antibodies, protein A, protein G or protein L.
25. The method of claim 1, wherein the method comprises conducting
a competitive binding assay.
26. The method of claim 1, wherein the components combined in step
(a) further comprise (xvi) a first detection reagent that competes
with the first analyte for binding to the first binding reagent and
(xvii) a second detection reagent that competes with the second
analyte for binding to the second binding reagent.
27. The method of claim 25, wherein the first and second detection
reagents comprise detectable labels.
28. The method of claim 25, wherein the first and second binding
reagents are antibodies and the first and second detection reagents
are structural analogs of the analytes.
29. The method of claim 1, wherein the measuring step (c) further
comprises measuring the presence of the first and second detectable
labels via optical absorbance, fluorescence, phosphorescence,
chemiluminescence, electrochemiluminescence, light scattering, or
magnetism.
30. The method of claim 29, wherein the first and second detectable
label is an electrochemiluminescent label and the measuring step
(c) further comprises measuring an electrochemiluminescent signal
and correlating the signal with an amount of first and second
analyte in the sample.
31. The method of claim 1, wherein the first and second binding
domains are positioned on an electrode and the measuring step
further comprises applying a voltage waveform to the electrode to
generate electrochemiluminescence.
32. The method of claim 1, wherein each of the first and second
binding domains is an element of an array of binding domains.
33. The method of claim 32, wherein the array is located within a
well of a multi-well plate.
34. The method of claim 1, wherein each of the first and second
binding domains are each positioned on a surface of one or more
microparticles.
35. The method of claim 34, wherein the particles are coded to
allow for identification of specific particles and discrimination
between the first and second binding domains.
36. The method of claim 1, wherein (i) the first targeting agent
and first targeting agent complement and (ii) the second targeting
agent and second targeting agent complement each comprise a
complementary oligonucleotide pair.
37. The method of claim 36, wherein the first and second binding
reagents are each antibodies.
38. The method of claim 36, wherein the complementary
oligonucleotide pair positioned on each of the first and second
binding domains is different and selected from: TABLE-US-00043 Pair
Sequence 1 acatcggtagtt (SEQ ID NO: 1 aactaccgatgt (SEQ ID NO: 2) 3
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
39. The method of claim 38, wherein there are at least 7 binding
domains.
40. The method of claim 38, wherein there are at least 10 binding
domains.
41. The method of claim 38, wherein there are at least 16 binding
domains.
42. The method of claim 38, wherein there are at least 25 binding
domains.
43. The method of claim 1, wherein each of the first and second
complementary targeting agents selectively bind to one of the first
and second binding domains, respectively.
44. The method of claim 43, wherein the cross-reactivity for
binding of the complementary targeting agent to other binding
domains is less than 5% of the binding to the one of the binding
domains.
45. The method of claim 43, wherein the cross-reactivity for
binding of each first and second complementary targeting agent to a
third binding domain is less than 1% of the binding to the one of
the binding domains.
46. The method of claim 43, wherein the cross-reactivity for
binding of each first and second complementary targeting agent to a
third binding domain is less than 0.5% of the binding to the one of
the binding domains.
47. The method of claim 43, wherein the cross-reactivity for
binding of each first and second complementary targeting agent to a
third binding domain is less than 0.1% of the binding to the one of
the binding domains.
48. The method of claim 1, wherein the first and second binding
reagents each bind different analytes of interest.
49. The method of claim 1, wherein the first and second binding
reagents are each preferentially selective for a different analyte
of interest.
50. The method of claim 1, wherein the first and second binding
reagents differ in the affinity and/or selectivity for different
analytes of interest.
51. The method of claim 43, wherein the cross-reactivity of the
first analyte to the second binding reagent is less than 5% of the
binding to the first binding reagent.
52. The method of claim 43, wherein the cross-reactivity of the
first analyte to the second binding reagent is less than 1% of the
binding to the first binding reagent.
53. The method of claim 43, wherein the cross-reactivity of the
first analyte to the second binding reagent is less than 0.5% of
the binding to the first binding reagent.
54. The method of claim 43, wherein the cross-reactivity of the
first analyte to the second binding reagent is less than 0.1% of
the binding to the first binding reagent.
55. The method of claim 43, wherein the observed cross-reactivity
of an analyte of interest for non-specific binding reagents is less
than 5% of the binding to the binding reagent selected for binding
to that analyte.
56. The method of claim 43, wherein the observed cross-reactivity
of an analyte of interest for non-specific binding reagents is less
than 1% of the binding to the binding reagent selected for binding
to that analyte.
57. The method of claim 43, wherein the observed cross-reactivity
of an analyte of interest for non-specific binding reagents is less
than 0.5% of the binding to the binding reagent selected for
binding to that analyte.
58. The method of claim 43, wherein the observed cross-reactivity
of an analyte of interest for non-specific binding reagents is less
than 0.1% of the binding to the binding reagent selected for
binding to that analyte.
59. The method of claim 1, wherein the first and second targeting
agents and first and second targeting agent complements,
respectively, are used to map a set of binding reagents to a set of
binding domains and each of the binding reagents in the set bind to
a different analyte of interest.
60. The method of claim 59 wherein each of the binding reagents in
the set is preferentially selective for a different analyte of
interest.
61. The method of claim 59 wherein each of the binding reagents in
the set differ in the affinity and/or selectivity for different
analytes of interest.
62. The method of claim 43, wherein the cross-reactivity of the
first analyte to a binding reagent located in the second binding
domain is less than 5% of the binding to a binding reagent located
in the first binding domain.
63. The method of claim 43, wherein the cross-reactivity of the
first analyte to a binding reagent located in the second binding
domain is less than 1% of the binding to a binding reagent located
in the first binding domain.
64. The method of claim 43, wherein the cross-reactivity of the
first analyte to a binding reagent located in the second binding
domain is less than 0.5% of the binding to a binding reagent
located in the first binding domain.
65. The method of claim 43, wherein the cross-reactivity of the
first analyte to a binding reagent located in the second binding
domain is less than 0.1% of the binding to a binding reagent
located in the first binding domain.
66. The method of claim 43, wherein the observed cross-reactivity
of an analyte of interest for binding reagents located in a
non-specific binding domain is less than 5% of the binding to the
binding reagents in the binding domain assigned to that
analyte.
67. The method of claim 43, wherein the observed cross-reactivity
of an analyte of interest for binding reagents located in a
non-specific binding domain is less than 1% of the binding to the
binding reagents in the binding domain assigned to that
analyte.
68. The method of claim 43, wherein the observed cross-reactivity
of an analyte of interest for binding reagents located in a
non-specific binding domain is less than 0.5% of the binding to the
binding reagents in the binding domain assigned to that
analyte.
69. The method of claim 43, wherein the observed cross-reactivity
of an analyte of interest for binding reagents located in a
non-specific binding domain is less than 0.1% of the binding to the
binding reagents in the binding domain assigned to that
analyte.
70. An oligonucleotide selected from: TABLE-US-00044 Sequence
acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
71. A kit comprising, in separate vials, at least 4
oligonucleotides, each comprising a different sequence selected
from: TABLE-US-00045 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
72. The kit of claim 71 wherein the kit comprises at least 7
oligonucleotides.
73. The kit of claim 71 wherein the kit comprises at least 10
oligonucleotides.
74. The kit of claim 71 wherein the kit comprises at least 16
oligonucleotides.
75. The kit of claim 71 wherein the kit comprises at least 25
oligonucleotides.
76. The kit of claim 71, wherein each of the oligonucleotides is
modified with a linking agent comprising a biotin, streptavidin,
avidin, amino group, thiol group, aldehyde group, hydrazide group,
azide group, alkyne group, maleimide group or iodoacetamide
group.
77. The kit of claim 71, wherein each of the at least 4
oligonucleotides are coupled to a different antibody.
78. The kit of claim 71, wherein each different sequence is not a
complement of an additional different sequence of the at least 4
oligonucleotides.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims benefit of U.S. Provisional
Application No. 62/047,097, filed Sep. 8, 2014, the entire contents
of which are incorporated herein by reference. Reference is made to
U.S. Provisional Application Ser. Nos. 61/775,860 and 61/778,727,
filed Mar. 11, 2013 and Mar. 13, 2013, respectively, the
disclosures of each are incorporated herein by reference in its
entirety. Reference is also made to the following U.S. application
Ser. Nos. 62/013,823, 61/993,581; 14/206,284; 14/208,040, and
14/203,638, the disclosures of which are hereby incorporated herein
by reference.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The Sequence Listing in the ASCII text file, named as
31380_SequenceListing.txt of 10 KB, created on Sep. 8, 2015, and
submitted to the United States Patent and Trademark Office via
EFS-Web, is incorporated herein by reference.
FIELD OF THE INVENTION
[0003] Improved methods and products for conducting binding assays
are provided. These methods include the use of a linking agent
complex that enables the user to configure an assay to meet his/her
needs from a standard set of assay materials. The products and
methods of the invention greatly enhance productivity and
flexibility in assay development.
BACKGROUND OF THE INVENTION
[0004] A substantial body of literature has been developed
concerning techniques that employ binding reactions, e.g.,
antigen-antibody reactions, nucleic acid hybridization and
receptor-ligand reactions, for the sensitive measurement of
analytes of interest in samples. The high degree of specificity in
many biochemical binding systems has led to many assay methods and
systems of value in a variety of markets including basic research,
human and veterinary diagnostics, environmental monitoring and
industrial testing. The presence of an analyte of interest may be
measured by directly measuring the participation of the analyte in
a binding reaction. In some approaches, this participation may be
indicated through the measurement of an observable label attached
to one or more of the binding materials.
[0005] Commercially available assays are generally supplied in a
pre-set configuration, offering the user little or no flexibility
to evaluate target(s) that may be of unique interest to him or her.
Such commercial panels may include target analytes that are of
little or no interest and/or they may not include the desired
target analyte(s). Therefore, there is a need to provide the user a
flexible method to configure a user-defined multiplexed assay using
a standard set of assay materials and methods.
SUMMARY OF THE INVENTION
[0006] The present invention contemplates the following specific
embodiments. Various modifications, additions and alterations may
be made to embodiments described herein by one skilled in the art
without departing from the spirit and scope of the invention. Such
modifications, additions, and alterations are intended to fall
within the scope of the claims.
[0007] Embodiment (1): a method of conducting a multiplexed binding
assay for a plurality of analytes of interest comprising:
[0008] combining, in one or more steps, the following components: a
sample comprising a first analyte of interest and a second analyte
of interest, a first targeting agent immobilized on a first binding
domain, a first targeting agent complement connected to a linking
agent, wherein the first targeting agent complement is a binding
partner of the first targeting agent, a first binding reagent
connected to a supplemental linking agent, wherein the first
binding reagent is a binding partner of the first analyte, a second
targeting agent immobilized on a second binding domain, a second
targeting agent complement connected to a linking agent, wherein
the second targeting agent complement is a binding partner of the
second targeting agent, a second binding reagent connected to a
supplemental linking agent, wherein the second binding reagent is a
binding partner of the second analyte, and optionally, at least two
copies of a bridging agent, wherein, if the bridging agent is
omitted, each linking agent is a binding partner of the
supplemental linking agent, or if the bridging agent is included,
the bridging agent has a first binding site for one of the linking
agents and an additional binding site for one of the supplemental
linking agents;
[0009] forming: a first binding complex on the first binding domain
comprising the first targeting agent, the first targeting agent
complement, the first binding reagent and the first analyte, and a
second binding complex on the second binding domain comprising the
second targeting agent, the second targeting agent complement, the
second binding reagent and the second analyte, and
[0010] measuring the amount of the first and second analytes on the
first and second binding domains, respectively.
[0011] In one example of embodiment (1), referred to as embodiment
(1)(a), the sample contains one or more additional analytes of
interest and for each additional analyte of interest, the combining
step further comprises combining, in one or more steps, an
additional targeting agent immobilized on an additional binding
domain, an additional targeting agent complement connected to a
linking agent, and an additional binding reagent connected to a
supplemental linking agent, and an additional binding complex on
the additional binding domain comprising the additional targeting
agent, the additional targeting agent complement, the additional
binding reagent and the additional analyte; the forming step
further comprises forming an additional binding complex on the
additional binding domain comprising the additional targeting
agent, the additional targeting agent complement, the additional
binding reagent and the additional analyte; and the measurement
further comprises measuring the amount of the additional analyte on
the additional binding domain.
[0012] In a further example of embodiment (1), referred to as
embodiment (1)(b), the first targeting agent complement and the
first binding reagent are provided as a pre-bound first targeting
complex comprising the first targeting agent complement and the
first binding reagent linked through a binding interaction between
the linking agent and supplemental linking agent; and the second
targeting agent complement and the second binding reagent are
provided as a pre-bound second targeting complex comprising the
second targeting agent complement and the second binding reagent
linked through a binding interaction between the linking agent and
supplemental linking agent.
[0013] Moreover, in another example of embodiment (1), referred to
as embodiment (1)(c), the first targeting complex is provided
pre-bound to the first targeting agent immobilized on the first
binding domain; and the second targeting complex is provided
pre-bound to the second targeting agent immobilized on the second
binding domain. In this example, combining step further includes:
combining the first and second targeting complexes with the sample
to form a mixture thereof, binding the first analyte to the first
binding reagent in the first targeting complex and binding the
second analyte to the second binding reagent in the second
targeting complex, contacting a mixture of the first and second
targeting complexes bound to first and second analytes,
respectively, with the first and second binding domains; and
binding the first targeting complex to the first targeting agent on
the first binding domain and binding the second targeting complex
to the second targeting agent on the second binding domain.
[0014] In particular example of embodiment (1), referred to as
embodiment (1)(d), the combining step further includes combining,
in a first volume of liquid, said first targeting agent complement,
said first binding reagent and, if used, said bridging reagent and
linking said first targeting agent complement and said first
binding reagent through their attached linking agents to form a
first targeting complex; and combining, in a second volume of
liquid, said second targeting agent complement, said second binding
reagent and, if used, said bridging reagent and linking said second
targeting agent complex complement and said second binding reagent
through their attached linking agents to form a second targeting
complex.
[0015] In a specific example of embodiment (1), referred to as
embodiment (1)(e), the combining step further includes combining
said first and second targeting complexes, contacting the
combination of said first and second targeting complexes with said
first and second binding domains, and binding said first targeting
complex to said first targeting agent on said first binding domain
and binding said second targeting complex to said second targeting
agent on said second binding domain. In this example, the combining
step further includes: combining the first and second targeting
complexes with the sample to form a mixture thereof, binding the
first analyte to the first binding reagent in the first targeting
complex and binding the second analyte to the second binding
reagent in the second targeting complex, contacting a mixture of
the first and second targeting complexes bound to first and second
analytes, respectively, with the first and second binding domains;
and binding the first targeting complex to the first targeting
agent on the first binding domain and binding the second targeting
complex to the second targeting agent on the second binding
domain.
[0016] In embodiments (1)(c) and (1)(e), the first and second
targeting complexes can be combined with the sample prior to
contacting the first and second targeting complexes with the first
and second binding domains. Moreover, in these specific
embodiments, the first and second targeting complexes can be
combined with the sample after contacting the first and second
targeting complexes with the first and second binding domains; or
the first and second targeting complexes can be combined with the
sample and contacted with the first and second binding domains at
the same time.
[0017] In embodiment (1)(d), the combining step can further include
the steps of: contacting said first and second targeting complexes
on said first and second binding domains with said sample, binding
said first analyte to the first binding reagent in said first
targeting complex and binding said second analyte to said second
binding reagent in said second targeting complex.
[0018] In embodiments (1) and (1)(a)-(e), the bridging agent can be
omitted and the linking agent can be bound to the supplemental
linking agent through a binding interaction between (a) a thiol
group and a maleimide or iodoacetamide groups; (b) an aldehyde and
a hydrazide; or (c) an alkyne and an azide.
[0019] In embodiments (1) and (1)(a)-(e), the bridging agent can be
omitted and (a) the linking agent is biotin and supplemental
linking agent is streptavidin or avidin; (b) the linking agent is
streptavidin or avidin and the supplemental linking agent is
biotin; (c) the linking agent is a peptide and the supplemental
linking agent is an anti-peptide antibody; or (d) the linking agent
is an anti-peptide antibody and the supplemental linking agent is a
peptide.
[0020] Alternatively or additionally, in embodiments (1) and
(1)(a)-(e), the bridging agent can be included, the bridging agent
can be streptavidin or avidin, and the linking agents and the
supplemental linking agents can each comprise biotin.
[0021] The first and second binding reagents referenced in
embodiments (1) and (1)(a)-(e), each can comprise a receptor,
ligand, antibody, hapten, antigen, epitope, mimitope, aptamer, or
an intercalater capable of binding to said first and second
analytes, respectively. For example, the first and second binding
reagents are antigens. Alternatively or additionally, the first and
second binding reagents are antibodies.
[0022] In embodiments (1) and (1)(a)-(e), the method can include
conducting a sandwich binding assay.
[0023] In one specific example of embodiments (1) and (1)(a)-(e),
the components combined in the combining step further comprise a
first detection reagent that binds the first analyte and a second
detection reagent that binds the second analyte, and the first and
second complexes formed in the second step further comprise the
first and second detection reagents, respectively. For example, the
first and second detection reagents each comprise a detectable
label. In another example of this embodiment, the first and second
binding reagents and the first and second detection reagents are
antibodies. Moreover, the first and second detection reagents can
bind to either the first or second analyte, and optionally, the
first and second detection reagents each comprise a detectable
label. In a specific example, the first and second binding reagents
are antigens, the analytes are antibodies against the antigens and
the detection reagents comprise anti-immunoglobulin antibodies,
protein A, protein G or protein L.
[0024] The method of embodiments (1) and (1)(a)-(e), can include
conducting a competitive binding assay.
[0025] Still further, the components combined in embodiments (1)
and (1)(a)-(e), can further comprise a first detection reagent that
competes with the first analyte for binding to the first binding
reagent and a second detection reagent that competes with the
second analyte for binding to the second binding reagent.
[0026] In embodiments (1) and (1)(a)-(e), one or more of the
following additional elements of the method are included: the first
and second detection reagents can comprise detectable labels; the
first and second binding reagents are antibodies and the first and
second detection reagents are structural analogs of the analytes;
the measuring step can comprise measuring the presence of the first
and second detectable labels via optical absorbance, fluorescence,
phosphorescence, chemiluminescence, electrochemiluminescence, light
scattering, or magnetism; the first and second detectable label is
an electrochemiluminescent label and the measuring step further
comprises measuring an electrochemiluminescent signal and
correlating the signal with an amount of first and second analyte
in the sample; the first and second binding domains are positioned
on an electrode and the measuring step further comprises applying a
voltage waveform to the electrode to generate
electrochemiluminescence; each of the first and second binding
domains is an element of an array of binding domains, and
optionally, the array is located within a well of a multi-well
plate; each of the first and second binding domains are each
positioned on a surface of one or more microparticles, and
optionally, the particles are coded to allow for identification of
specific particles and discrimination between the first and second
binding domains.
[0027] Embodiment (1)(f): includes the elements of embodiment (1)
and one or more additional features of embodiment (1)(a)-(e),
wherein the first targeting agent and first targeting agent
complement and the second targeting agent and second targeting
agent complement each comprise a complementary oligonucleotide
pair. In this specific example, the first and second binding
reagents can each be antibodies. Moreover, in this specific
example, the complementary oligonucleotide pair positioned on each
of the first and second binding domains is different and selected
from:
TABLE-US-00001 pair # Sequence (5'-3') 1 Acatcggtagtt (SEQ ID NO:
1) Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0028] Embodiment (1)(g): includes the elements of embodiment (1)
and one or more additional features of embodiment (1)(a)-(e),
wherein the first targeting agent and first targeting agent
complement and the second targeting agent and second targeting
agent complement each comprise a complementary oligonucleotide
pair. In this specific example, the first and second binding
reagents can each be antibodies. Moreover, in this specific
example, the complementary oligonucleotide pair positioned on each
of the first and second binding domains is different and selected
from:
TABLE-US-00002 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0029] In embodiments (1)(f) and/or (1)(g), there are at least 7
binding domains, or at least 10 binding domains; or at least 16
binding domains; or at least 25 binding domains.
[0030] In embodiments (1) and (1)(a), each of the first and second
complementary targeting agents can selectively bind to one of the
first and second binding domains, respectively. For example, the
cross-reactivity for binding of the complementary targeting agent
to other binding domains is less than 5% of the binding to the one
of the binding domains; or the cross-reactivity for binding of each
first and second complementary targeting agent to a third binding
domain is less than 1% of the binding to the one of the binding
domains; or the cross-reactivity for binding of each first and
second complementary targeting agent to a third binding domain is
less than 0.5% of the binding to the one of the binding domains; or
the cross-reactivity for binding of each first and second
complementary targeting agent to a third binding domain is less
than 0.1% of the binding to the one of the binding domains.
[0031] In embodiments (1) and (1)(a), the first and second binding
reagents can each bind different analytes of interest; or the first
and second binding reagents are each preferentially selective for a
different analyte of interest.
[0032] The first and second binding reagents of any one of the
foregoing embodiments can differ in the affinity and/or selectivity
for different analytes of interest, e.g., the cross-reactivity of
the first analyte to the second binding reagent is less than 5% of
the binding to the first binding reagent, or the cross-reactivity
of the first analyte to the second binding reagent is less than 1%
of the binding to the first binding reagent, or the
cross-reactivity of the first analyte to the second binding reagent
is less than 0.5% of the binding to the first binding reagent, or
the cross-reactivity of the first analyte to the second binding
reagent is less than 0.1% of the binding to the first binding
reagent, or the observed cross-reactivity of an analyte of interest
for non-specific binding reagents is less than 5% of the binding to
the binding reagent selected for binding to that analyte, or the
observed cross-reactivity of an analyte of interest for
non-specific binding reagents is less than 1% of the binding to the
binding reagent selected for binding to that analyte, or the
observed cross-reactivity of an analyte of interest for
non-specific binding reagents is less than 0.5% of the binding to
the binding reagent selected for binding to that analyte, or the
observed cross-reactivity of an analyte of interest for
non-specific binding reagents is less than 0.1% of the binding to
the binding reagent selected for binding to that analyte. Still
further in this specific example of the foregoing embodiments, the
cross-reactivity of the first analyte to a binding reagent located
in the second binding domain is less than 5% of the binding to a
binding reagent located in the first binding domain, or the
cross-reactivity of the first analyte to a binding reagent located
in the second binding domain is less than 1% of the binding to a
binding reagent located in the first binding domain, or the
cross-reactivity of the first analyte to a binding reagent located
in the second binding domain is less than 0.5% of the binding to a
binding reagent located in the first binding domain, or the
cross-reactivity of the first analyte to a binding reagent located
in the second binding domain is less than 0.1% of the binding to a
binding reagent located in the first binding domain, or the
observed cross-reactivity of an analyte of interest for binding
reagents located in a non-specific binding domain is less than 5%
of the binding to the binding reagents in the binding domain
assigned to that analyte, or the observed cross-reactivity of an
analyte of interest for binding reagents located in a non-specific
binding domain is less than 1% of the binding to the binding
reagents in the binding domain assigned to that analyte, or the
observed cross-reactivity of an analyte of interest for binding
reagents located in a non-specific binding domain is less than 0.5%
of the binding to the binding reagents in the binding domain
assigned to that analyte, or the observed cross-reactivity of an
analyte of interest for binding reagents located in a non-specific
binding domain is less than 0.1% of the binding to the binding
reagents in the binding domain assigned to that analyte.
[0033] In embodiments (1) and (1)(a), the first and second
targeting agents and first and second targeting agent complements,
respectively, can be used to map a set of binding reagents to a set
of binding domains and each of the binding reagents in the set bind
to a different analyte of interest. For example, each of the
binding reagents in the set is preferentially selective for a
different analyte of interest, or each of the binding reagents in
the set differ in the affinity and/or selectivity for different
analytes of interest.
[0034] Embodiment (2): an oligonucleotide selected from:
TABLE-US-00003 Acatcggtagtt (SEQ ID NO: 1) Aactaccgatgt (SEQ ID NO:
2) acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4)
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)
aggttcagtgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8)
atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10)
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28)
ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
tacccggaataa (SEQ ID NO: 41) ttattccgggta (SEQ ID NO: 42)
tgcttgacttgg (SEQ ID NO: 43) ccaagtcaagca (SEQ ID NO: 44)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
ttgtctagcggc (SEQ ID NO: 47) gccgctagacaa (SEQ ID NO: 48)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
[0035] Embodiment (3): an oligonucleotide selected from:
TABLE-US-00004 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0036] Embodiment (4): a kit comprising, in separate vials, at
least 4 oligonucleotides, each comprising a different sequence
selected from:
TABLE-US-00005 Acatcggtagtt (SEQ ID NO: 1) Aactaccgatgt (SEQ ID NO:
2) acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4)
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)
aggttcagtgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8)
atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10)
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28)
ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
tacccggaataa (SEQ ID NO: 41) ttattccgggta (SEQ ID NO: 42)
tgcttgacttgg (SEQ ID NO: 43) ccaagtcaagca (SEQ ID NO: 44)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
ttgtctagcggc (SEQ ID NO: 47) gccgctagacaa (SEQ ID NO: 48)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
[0037] Embodiment (5): a kit comprising, in separate vials, at
least 4 oligonucleotides, each comprising a different sequence
selected from:
TABLE-US-00006 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0038] In the kit of embodiments (4) and (5), the kit can comprise
at least 7 oligonucleotides, or at least 10 oligonucleotides, or at
least 16 oligonucleotides, or at least 25 oligonucleotides.
Moreover, each of the oligonucleotides of these specific
embodiments can be modified with a linking agent comprising a
biotin, streptavidin, avidin, amino group, thiol group, aldehyde
group, hydrazide group, azide group, alkyne group, maleimide group
or iodoacetamide group. In another specific example of embodiments
(4) and (5), each of the at least 4 oligonucleotides can be coupled
to a different antibody, and optionally, each different sequence is
not a complement of an additional different sequence of the at
least 4 oligonucleotides.
[0039] Embodiment (6): a mixture comprising at least 4
oligonucleotides, each comprising a different sequence selected
from:
TABLE-US-00007 Acatcggtagtt (SEQ ID NO: 1) Aactaccgatgt (SEQ ID NO:
2) acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4)
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)
aggttcagtgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8)
atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10)
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28)
ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
tacccggaataa (SEQ ID NO: 41) ttattccgggta (SEQ ID NO: 42)
tgcttgacttgg (SEQ ID NO: 43) ccaagtcaagca (SEQ ID NO: 44)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
ttgtctagcggc (SEQ ID NO: 47) gccgctagacaa (SEQ ID NO: 48)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
[0040] wherein each of the different sequences are coupled to a
different antibody.
[0041] Embodiment (7): a mixture comprising at least 4
oligonucleotides, each comprising a different sequence selected
from:
TABLE-US-00008 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0042] wherein each of the different sequences are coupled to a
different antibody.
[0043] In embodiments (6) and (7), the mixture can include at least
7 oligonucleotides, or at least 10 oligonucleotides; or at least 16
oligonucleotides; or at least 25 oligonucleotides. Moreover, in a
specific example of embodiments (6) and (7), each different
sequence is not a complement of an additional different sequence of
the at least 4 oligonucleotides.
[0044] Embodiment (8): an array comprising a plurality of at least
4 binding domains, wherein one or more, and optionally, each
binding domain has immobilized thereon a different oligonucleotide
sequence selected from:
TABLE-US-00009 Acatcggtagtt (SEQ ID NO: 1) Aactaccgatgt (SEQ ID NO:
2) acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4)
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)
aggttcagtgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8)
atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10)
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28)
ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
tacccggaataa (SEQ ID NO: 41) ttattccgggta (SEQ ID NO: 42)
tgcttgacttgg (SEQ ID NO: 43) ccaagtcaagca (SEQ ID NO: 44)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
ttgtctagcggc (SEQ ID NO: 47) gccgctagacaa (SEQ ID NO: 48)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
[0045] Embodiment (9): an array comprising a plurality of at least
4 binding domains, wherein one or more and optionally, each binding
domain has immobilized thereon a different oligonucleotide sequence
selected from:
TABLE-US-00010 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0046] In embodiments (8) and (9), the array can comprise at least
7 binding domains, or at least 10 binding domains, or at least 16
binding domains, or at least 25 binding domains, and optionally,
each different sequence is not a complement of an additional
different sequence of the at least 4 oligonucleotides
[0047] Embodiment (10): a multi-well plate having one or more
copies of an oligonucleotide array within at least one well(s) of
the plate, the array is positioned on a plurality of binding
domains, wherein one or more and optionally, at least 4 of the
binding domains have immobilized thereon a different
oligonucleotide sequence selected from:
TABLE-US-00011 Acatcggtagtt (SEQ ID NO: 1) Aactaccgatgt (SEQ ID NO:
2) acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4)
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)
aggttcagtgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8)
atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10)
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28)
ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
tacccggaataa (SEQ ID NO: 41) ttattccgggta (SEQ ID NO: 42)
tgcttgacttgg (SEQ ID NO: 43) ccaagtcaagca (SEQ ID NO: 44)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
ttgtctagcggc (SEQ ID NO: 47) gccgctagacaa (SEQ ID NO: 48)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
[0048] Embodiment (11): a multi-well plate having one or more
copies of an oligonucleotide array within at least one well(s) of
the plate, the array is positioned on a plurality of binding
domains, wherein one or more and optionally, at least 4 of the
binding domains have immobilized thereon a different
oligonucleotide sequence selected from:
TABLE-US-00012 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0049] The plate of embodiments (10) and (11) can include at least
24 wells, or at least 96 wells, or at least 384 wells, and/or
optionally, the array comprises at least 7 oligonucleotides, or at
least 10 oligonucleotides, or at least 16 oligonucleotides, or at
least 25 oligonucleotides, wherein optionally, each different
sequence is not a complement of an additional different sequence of
the at least 4 oligonucleotides
[0050] Embodiment (12): a kit comprising a set of at least 4
microparticle reagents, in one or more vials, wherein each
microparticle reagent of the set comprises a microparticle having
immobilized thereon a different oligonucleotide sequence selected
from:
TABLE-US-00013 Acatcggtagtt (SEQ ID NO: 1) Aactaccgatgt (SEQ ID NO:
2) acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4)
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)
aggttcagtgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8)
atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10)
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28)
ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
tacccggaataa (SEQ ID NO: 41) ttattccgggta (SEQ ID NO: 42)
tgcttgacttgg (SEQ ID NO: 43) ccaagtcaagca (SEQ ID NO: 44)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
ttgtctagcggc (SEQ ID NO: 47) gccgctagacaa (SEQ ID NO: 48)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
[0051] Embodiment (13): a kit comprising a set of at least 4
microparticle reagents, in one or more vials, wherein each
microparticle reagent of the set comprises a microparticle having
immobilized thereon a different oligonucleotide sequence selected
from:
TABLE-US-00014 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0052] The kit of embodiments (12) and (13) can include a set of at
least 7 microparticles, or a set of at least 10 microparticles, or
a set of at least 16 microparticles, or a set of at least 25
microparticles, and/or optionally, each different sequence is not a
complement of an additional different sequence of the at least 4
oligonucleotides.
[0053] The microparticles of embodiments (12) and (13) can be coded
and the different microparticle reagents can have different
identifying codes.
[0054] Embodiment (14): a kit comprising a multi-well plate having
one or more copies of an oligonucleotide array within at least one
well(s) of the plate, the array is positioned on a plurality of
binding domains, wherein one or more and optionally, at least 4 of
the binding domains have immobilized thereon a different
oligonucleotide sequence selected from:
TABLE-US-00015 Acatcggtagtt (SEQ ID NO: 1) Aactaccgatgt (SEQ ID NO:
2) acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4)
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)
aggttcagtgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8)
atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10)
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28)
ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
tacccggaataa (SEQ ID NO: 41) ttattccgggta (SEQ ID NO: 42)
tgcttgacttgg (SEQ ID NO: 43) ccaagtcaagca (SEQ ID NO: 44)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
ttgtctagcggc (SEQ ID NO: 47) gccgctagacaa (SEQ ID NO: 48)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
[0055] and [0056] (i) a set of oligonucleotides comprised of two or
more oligonucleotides selected from:
TABLE-US-00016 [0056] Acatcggtagtt (SEQ ID NO: 1) Aactaccgatgt (SEQ
ID NO: 2) acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4)
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)
aggttcagtgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8)
atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10)
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28)
ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
tacccggaataa (SEQ ID NO: 41) ttattccgggta (SEQ ID NO: 42)
tgcttgacttgg (SEQ ID NO: 43) ccaagtcaagca (SEQ ID NO: 44)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
ttgtctagcggc (SEQ ID NO: 47) gccgctagacaa (SEQ ID NO: 48)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
[0057] wherein the oligonucleotides in component (ii) are
complementary to the oligonucleotides in component (i).
[0058] Embodiment (15): a kit comprising a multi-well plate having
one or more copies of an oligonucleotide array within at least one
well(s) of the plate, the array is positioned on a plurality of
binding domains, wherein one or more and optionally, at least 4 of
the binding domains have immobilized thereon a different
oligonucleotide sequence selected from:
TABLE-US-00017 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0059] and [0060] (i) a set of oligonucleotides comprised of two or
more oligonucleotides selected from:
TABLE-US-00018 [0060] Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0061] wherein the oligonucleotides in component (ii) are
complementary to the oligonucleotides in component (i).
[0062] Embodiment (16): a method of conducting a binding assay for
a plurality of analytes comprising: (a) contacting a sample with
two or more binding domains linked to at least a first and second
binding reagent that each bind a first and second analyte,
respectively, of the plurality of analytes to form complexes
comprising the first analyte bound to the first binding reagent and
the second analyte bound to the second binding reagent, wherein (x)
the first binding domain comprises a first binding reagent complex
comprising (i) a first targeting agent bound to the first binding
domain and to a first targeting agent complement; and (ii) the
first binding reagent bound to the first targeting agent complement
via a linking complex; and (y) the second binding domain comprises
a second binding reagent complex comprising (i) a second targeting
agent bound to the second binding domain and to a second targeting
agent complement; and (ii) the second binding reagent bound to the
second targeting agent complement via a linking complex; (b)
contacting the first and second binding reagent complexes with a
plurality of detection reagents comprising a first detection
reagent that binds the first analyte or a complex comprising the
first analyte, and a second detection reagent that binds the second
analyte or a complex comprising the second analyte; and (c)
measuring the amount of the first and second analytes bound to the
two or more binding domains.
[0063] In embodiment (16), one or more of the following features
can be adopted: the first and second binding reagents each comprise
a receptor, ligand, antibody, hapten, antigen, epitope, mimitope,
aptamer, or an intercalater capable of binding to the first and
second analyte, respectively, e.g., the first and second binding
reagents each comprise an antibody capable of binding to the first
and second analyte, respectively; the first targeting agent and the
first targeting agent complement comprise an oligonucleotide and a
complementary oligonucleotide, a receptor-ligand pair, an
antigen-antibody pair, a hapten-antibody pair, an epitope-antibody
pair, an mimitope-antibody pair, an aptamer-target molecule pair, a
hybridization partners, or an intercalater-target molecule pair;
the second targeting agent and the second targeting agent
complement comprise an oligonucleotide and a complementary
oligonucleotide, a receptor-ligand pair, an antigen-antibody pair,
a hapten-antibody pair, an epitope-antibody pair, an
mimitope-antibody pair, an aptamer-target molecule pair, a
hybridization partners, or an intercalater-target molecule pair;
the first targeting agent and the first targeting agent complement
comprise an oligonucleotide and a complementary oligonucleotide,
respectively; the second targeting agent and the second targeting
agent complement comprise an oligonucleotide and a complementary
oligonucleotide, respectively; the linking complex comprises a
linking agent and a supplemental linking agent connected thereto,
e.g., the linking complex is formed by a binding interaction
between (a) a thiol group and a maleimide or iodoacetamide; (b) an
aldehyde and a hydrazide; or (c) an alkyne and an azide; or the (a)
the linking agent is biotin and supplemental linking agent is
streptavidin or avidin; (b) the linking agent is streptavidin or
avidin and the supplemental linking agent is biotin; (c) the
linking agent is a peptide and the supplemental linking agent is an
anti-peptide antibody; or (d) the linking agent is an anti-peptide
antibody and the supplemental linking agent is a peptide.
[0064] Moreover, embodiment (16) can optionally include one or more
of the following features: each of the plurality of detection
reagents comprise a detectable label; a subset of the plurality of
detection reagents comprise a detectable label; the measuring step
comprises measuring the presence of the detectable label in the
sample via optical absorbance, fluorescence, phosphorescence,
chemiluminescence, electrochemiluminescence, light scattering, or
magnetism; the detectable label is an electrochemiluminescent label
and the measuring step comprises measuring an
electrochemiluminescent signal and correlating the signal with an
amount of analyte in the sample, e.g., the two or more binding
domains are positioned on an electrode and the measuring step
further comprises applying a voltage waveform to the electrode to
generate electrochemiluminescence, and optionally, the two or more
binding domains are located within one or more wells of a
multi-well plate.
[0065] Embodiment (16) can include conducting a sandwich
immunoassay or a competitive immunoassay.
[0066] In embodiment (16), the first targeting agent and the first
targeting agent complement can comprise a pair of oligonucleotides,
wherein the pair is selected from:
TABLE-US-00019 pair # Sequence (5'-3') 1 Acatcggtagtt (SEQ ID NO:
1) Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0067] Moreover, in embodiment (16), the first targeting agent and
the first targeting agent complement can comprise a pair of
oligonucleotides, wherein the pair is selected from:
TABLE-US-00020 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0068] In one example of embodiment (16), the second targeting
agent and the second targeting agent complement can include a pair
of oligonucleotides, wherein the pair is selected from:
TABLE-US-00021 pair # Sequence (5'-3') 1 Acatcggtagtt (SEQ ID NO:
1) Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0069] In a further example of embodiment (16), the second
targeting agent and the second targeting agent complement can
include a pair of oligonucleotides, wherein the pair is selected
from:
TABLE-US-00022 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0070] Embodiment (16) can include one or more of the following
elements: the first targeting agent and first targeting agent
complement comprise a first oligonucleotide and a first
complementary oligonucleotide, respectively, and the first
oligonucleotide and the first complementary oligonucleotide each
comprise approximately 10 to 50 bases; the first targeting agent
and first targeting agent complement comprise a first
oligonucleotide and a first complementary oligonucleotide,
respectively, and the first oligonucleotide and the first
complementary oligonucleotide each comprise approximately 10 to 25
bases; the first binding reagent is an antibody comprising a biotin
molecule and the first targeting agent is a first oligonucleotide
comprising a streptavidin molecule and the linking complex is
formed by a reaction between the biotin and streptavidin molecules;
and/or the second binding reagent is an antibody comprising an
additional biotin molecule and the second targeting agent is a
second oligonucleotide comprising an additional streptavidin
molecule and the linking complex is formed by a reaction between
the additional biotin and the additional streptavidin
molecules.
[0071] Embodiment (17): a method of conducting a binding assay for
a plurality of analytes comprising: (a) forming a first binding
reagent complex comprising a first binding reagent specific for a
first analyte in the plurality of analytes and a first targeting
agent, wherein the first binding reagent is bound to a linking
agent and the first targeting agent is bound to a supplemental
linking agent wherein the first binding reagent complex is formed
by a reaction between the linking agent and the supplemental
linking agent; (b) forming a second binding reagent complex
comprising a second binding reagent specific for a second analyte
in the plurality of analytes and a second targeting agent, wherein
the second binding reagent is bound to a second linking agent and
the second targeting agent is bound to a second linking agent
complement wherein the second binding reagent complex is formed by
a reaction between the second linking agent and the second linking
agent complement; (c) mixing the first and second binding reagent
complexes with two or more binding domains each linked to a first
targeting agent complement and a second targeting agent complement,
respectively, under conditions sufficient to bind the first
targeting agent to the first targeting agent complement and the
second targeting agent to the second targeting agent complement;
(d) mixing a sample comprising the plurality of analytes to the
mixture formed in step (c); (e) adding a plurality of additional
binding reagents to the mixture formed in step (d), wherein the
plurality of additional binding reagents includes (i) a first
detection reagent specific for the first analyte and/or a first
binding reagent-first analyte complex; and (ii) a second detection
reagent specific for the second analyte and/or a second binding
reagent-second analyte complex; and (f) measuring the amount of the
first and second analytes bound to the binding domains.
[0072] Embodiment (17) can include one or more of the following
features: the first and second binding reagents each comprise a
receptor, ligand, antibody, hapten, antigen, epitope, mimitope,
aptamer, or an intercalater capable of binding to the first and
second analytes, respectively, e.g., the first and second binding
reagents each comprise an antibody capable of binding to the first
and second analyte, respectively; the first targeting agent and the
first targeting agent complement comprise an oligonucleotide and a
complementary oligonucleotide, a receptor-ligand pair, an
antigen-antibody pair, a hapten-antibody pair, an antigen-antibody
pair, an epitope-antibody pair, an mimitope-antibody pair, an
aptamer-target molecule pair, hybridization partners, or an
intercalater-target molecule pair; the second targeting agent and
the second targeting agent complement comprise an oligonucleotide
and a complementary oligonucleotide, a receptor-ligand pair, an
antigen-antibody pair, a hapten-antibody pair, an antigen-antibody
pair, an epitope-antibody pair, an mimitope-antibody pair, an
aptamer-target molecule pair, hybridization partners, or an
intercalater-target molecule pair; the first targeting agent and
the first targeting agent complement comprise an oligonucleotide
and a complementary oligonucleotide; the second targeting agent and
the second targeting agent complement comprise an oligonucleotide
and a complementary oligonucleotide; the linking complex comprises
a linking agent and a supplemental linking agent connected thereto,
e.g., the linking complex is formed by a binding interaction
between (a) a thiol group and a maleimide or iodoacetamide; (b) an
aldehyde and a hydrazide; or (c) an alkyne and an azide; the (a)
the linking agent is biotin and supplemental linking agent is
streptavidin or avidin; (b) the linking agent is streptavidin or
avidin and the supplemental linking agent is biotin; (c) the
linking agent is a peptide and the supplemental linking agent is an
anti-peptide antibody; or (d) the linking agent is an anti-peptide
antibody and the supplemental linking agent is a peptide.
[0073] Moreover, embodiment (17) can further include one or more of
the following features: each of the plurality of detection reagents
comprise a detectable label, e.g., a subset of the plurality of
detection reagents comprise a detectable label; the measuring step
comprises measuring the presence of the detectable label in the
sample via optical absorbance, fluorescence, phosphorescence,
chemiluminescence, electrochemiluminescence, light scattering, or
magnetism; the detectable label is an electrochemiluminescent label
and the measuring step comprises measuring an
electrochemiluminescent signal and correlating the signal with an
amount of analyte in the sample; the two or more binding domains
are positioned on an electrode and the measuring step further
comprises applying a voltage waveform to the electrode to generate
electrochemiluminescence, e.g., the two or more binding domains are
located within one or more wells of a multi-well plate.
[0074] Embodiment (17) can comprise conducting a sandwich
immunoassay or a competitive immunoassay.
[0075] In embodiment (17), the first targeting agent and the first
targeting agent complement comprise a pair of oligonucleotides,
wherein the pair is selected from:
TABLE-US-00023 pair # Sequence (5'-3') 1 Acatcggtagtt (SEQ ID NO:
1) Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0076] In a specific example of embodiment (17), the first
targeting agent and the first targeting agent complement comprise a
pair of oligonucleotides, wherein the pair is selected from:
TABLE-US-00024 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0077] In a further example of embodiment (17), the second
targeting agent and the second targeting agent complement comprise
a pair of oligonucleotides, wherein the pair is selected from:
TABLE-US-00025 pair # Sequence (5'-3') 1 Acatcggtagtt (SEQ ID NO:
1) Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0078] In yet a further example of embodiment (17), the second
targeting agent and the second targeting agent complement comprise
a pair of oligonucleotides, wherein the pair is selected from:
TABLE-US-00026 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0079] Embodiment (17) can include one or more of the following
elements: the first targeting agent and the first targeting agent
complement comprise a first oligonucleotide and a first
complementary oligonucleotide, respectively, and the first
oligonucleotide and the first complementary oligonucleotide each
comprise approximately 10 to 50 bases; the first targeting agent
and the first targeting agent complement comprise a first
oligonucleotide and a first complementary oligonucleotide,
respectively, and the first oligonucleotide and the first
complementary oligonucleotide each comprise approximately 10 to 25
bases; the first binding reagent is an antibody comprising a biotin
molecule and the first targeting agent comprises a first
oligonucleotide including a streptavidin molecule and the linking
complex is formed by a reaction between the biotin and streptavidin
molecules; the second binding reagent is an antibody comprising an
additional biotin molecule and the second targeting agent comprises
a second oligonucleotide including an additional streptavidin
molecule and the linking complex is formed by a reaction between
the additional biotin and the additional streptavidin molecules;
the second targeting agent and the second targeting agent
complement comprise a second oligonucleotide and a second
complementary oligonucleotide, respectively, and the second
oligonucleotide and the second complementary oligonucleotide each
comprise approximately 10 to 50 bases; the second targeting agent
and the second targeting agent complement comprise a second
oligonucleotide and a second complementary oligonucleotide,
respectively, and the second oligonucleotide and the second
complementary oligonucleotide each comprise approximately 10 to 25
bases; the second binding reagent is an antibody comprising a
biotin molecule and the second targeting agent comprises a second
oligonucleotide including a streptavidin molecule and the linking
complex is formed by a reaction between the biotin and streptavidin
molecules; and/or the method further comprises the step of washing
the mixture formed in step (c) prior to mixing step (d).
[0080] Embodiment (18): a method of conducting a binding assay for
a plurality of analytes in a sample comprising: (a) forming a first
binding reagent complex comprising a first binding reagent specific
for a first analyte in the plurality of analytes and a first
targeting agent, wherein the first binding reagent is bound to a
linking agent and the first targeting agent is bound to a
supplemental linking agent wherein the first binding reagent
complex is formed by a reaction between the linking agent and the
supplemental linking agent; (b) forming a second binding reagent
complex comprising a second binding reagent specific for a second
analyte in the plurality of analytes and a second targeting agent,
wherein the second binding reagent is bound to a second linking
agent and the second targeting agent is bound to a second linking
agent complement wherein the second binding reagent complex is
formed by a reaction between the second linking agent and the
second linking agent complement; (c) mixing the first and second
binding reagent complexes and the sample with two or more binding
domains each linked to a first targeting agent complement and a
second targeting agent complement, respectively, under conditions
sufficient to bind the first targeting agent to the first targeting
agent complement and the second targeting agent to the second
targeting agent complement; (d) adding a plurality of additional
binding reagents to the mixture formed in step (c), wherein the
plurality of additional binding reagents includes (i) a first
detection reagent specific for the first analyte and/or a first
binding reagent-first analyte complex; and (ii) a second detection
reagent specific for the second analyte and/or a second binding
reagent-second analyte complex; and (e) measuring the amount of the
first and second analytes bound to the binding domains.
[0081] In addition, embodiment (18) can include one or more of the
following: the first and second binding reagents each comprise a
receptor, ligand, antibody, hapten, antigen, epitope, mimitope,
aptamer, or an intercalater capable of binding to the first and
second analytes, respectively; first and second binding reagents
each comprise an antibody capable of binding to the first and
second analyte, respectively; first targeting agent and the first
targeting agent complement comprise an oligonucleotide and a
complementary oligonucleotide, a receptor-ligand pair, an
antigen-antibody pair, a hapten-antibody pair, an antigen-antibody
pair, an epitope-antibody pair, an mimitope-antibody pair, an
aptamer-target molecule pair, hybridization partners, or an
intercalater-target molecule pair; the second targeting agent and
the second targeting agent complement comprise an oligonucleotide
and a complementary oligonucleotide, a receptor-ligand pair, an
antigen-antibody pair, a hapten-antibody pair, an antigen-antibody
pair, an epitope-antibody pair, an mimitope-antibody pair, an
aptamer-target molecule pair, hybridization partners, or an
intercalater-target molecule pair; the first targeting agent and
the first targeting agent complement comprise an oligonucleotide
and a complementary oligonucleotide; the second targeting agent and
the second targeting agent complement comprise an oligonucleotide
and a complementary oligonucleotide; the linking complex comprises
a linking agent and a supplemental linking agent connected thereto,
e.g., the linking complex is formed by a binding interaction
between (a) a thiol group and a maleimide or iodoacetamide; (b) an
aldehyde and a hydrazide; or (c) an alkyne and an azide; e.g., (a)
the linking agent is biotin and supplemental linking agent is
streptavidin or avidin; (b) the linking agent is streptavidin or
avidin and the supplemental linking agent is biotin; (c) the
linking agent is a peptide and the supplemental linking agent is an
anti-peptide antibody; or (d) the linking agent is an anti-peptide
antibody and the supplemental linking agent is a peptide.
[0082] Still further, embodiment (18) can include one or more of
the following elements: each of the plurality of detection reagents
comprise a detectable label; the plurality of detection reagents
comprise a detectable label; the measuring step comprises measuring
the presence of the detectable label in the sample via optical
absorbance, fluorescence, phosphorescence, chemiluminescence,
electrochemiluminescence, light scattering, or magnetism; the
detectable label is an electrochemiluminescent label and the
measuring step comprises measuring an electrochemiluminescent
signal and correlating the signal with an amount of analyte in the
sample; the two or more binding domains are positioned on an
electrode and the measuring step further comprises applying a
voltage waveform to the electrode to generate
electrochemiluminescence; and/or the two or more binding domains
are located within one or more wells of a multi-well plate.
[0083] Embodiment (18) can include conducting a sandwich
immunoassay or a competitive immunoassay.
[0084] In a specific example of embodiment (18), the first
targeting agent and the first targeting agent complement comprise a
pair of oligonucleotides, wherein the pair is selected from:
TABLE-US-00027 pair # Sequence (5'-3') 1 Acatcggtagtt (SEQ ID NO:
1) Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0085] In a another example of embodiment (18), the first targeting
agent and the first targeting agent complement comprise a pair of
oligonucleotides, wherein the pair is selected from:
TABLE-US-00028 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 19)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0086] In yet another example of embodiment (18), the second
targeting agent and the second targeting agent complement comprise
a pair of oligonucleotides, wherein the pair is selected from:
TABLE-US-00029 pair # Sequence (5'-3') 1 Acatcggtagtt (SEQ ID NO:
1) Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0087] In a further example of embodiment (18), the second
targeting agent and the second targeting agent complement comprise
a pair of oligonucleotides, wherein the pair is selected from:
TABLE-US-00030 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0088] Embodiment (18) can include one or more of the following
features: the first targeting agent and the first targeting agent
complement comprise a first oligonucleotide and a first
complementary oligonucleotide, respectively, and the first
oligonucleotide and the first complementary oligonucleotide each
comprise approximately 10 to 50 bases; the first targeting agent
and the first targeting agent complement comprise a first
oligonucleotide and a first complementary oligonucleotide,
respectively, and the first oligonucleotide and the first
complementary oligonucleotide each comprise approximately 10 to 25
bases; the first binding reagent is an antibody comprising a biotin
molecule and the first targeting agent comprises a first
oligonucleotide including a streptavidin molecule and the linking
complex is formed by a reaction between the biotin and streptavidin
molecules; the second binding reagent is an antibody comprising an
additional biotin molecule and the second targeting agent comprises
a second oligonucleotide including an additional streptavidin
molecule and the linking complex is formed by a reaction between
the additional biotin and the additional streptavidin molecules;
the second targeting agent and the second targeting agent
complement comprise a second oligonucleotide and a second
complementary oligonucleotide, respectively, and the second
oligonucleotide and the second complementary oligonucleotide each
comprise approximately 10 to 50 bases; the second targeting agent
and the second targeting agent complement comprise a second
oligonucleotide and a second complementary oligonucleotide,
respectively, and the second oligonucleotide and the second
complementary oligonucleotide each comprise approximately 10 to 25
bases; the second binding reagent is an antibody comprising a
biotin molecule and the second targeting agent comprises a second
oligonucleotide including a streptavidin molecule and the linking
complex is formed by a reaction between the biotin and streptavidin
molecules; and/or the method further comprises the step of washing
the mixture formed in step (c) prior to mixing step (d).
[0089] Embodiment (19): a kit comprising: (a) a multi-well plate
comprising a plurality of discrete binding domains each comprising
a first and second oligonucleotide, respectively; (b) in a separate
vial, container, or compartment, a set of targeting reagents
comprising a first oligonucleotide complement bound to a linking
agent and a second oligonucleotide complement bound to a second
linking agent, wherein (i) the first oligonucleotide and the first
oligonucleotide complement comprise a first pair of
oligonucleotides, and (ii) the second oligonucleotide and the
second oligonucleotide complement comprise a second pair of
oligonucleotides, wherein the first and second pair of
oligonucleotides, respectively, are selected from:
TABLE-US-00031 pair # Sequence (5'-3') 1 Acatcggtagtt (SEQ ID NO:
1) Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0090] Embodiment (20): a kit comprising: (a) a multi-well plate
comprising a plurality of discrete binding domains each comprising
a first and second oligonucleotide, respectively; (b) in a separate
vial, container, or compartment, a set of targeting reagents
comprising a first oligonucleotide complement bound to a linking
agent and a second oligonucleotide complement bound to a second
linking agent, wherein (i) the first oligonucleotide and the first
oligonucleotide complement comprise a first pair of
oligonucleotides, and (ii) the second oligonucleotide and the
second oligonucleotide complement comprise a second pair of
oligonucleotides, wherein the first and second pair of
oligonucleotides, respectively, are selected from:
TABLE-US-00032 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0091] Embodiments (19) and (20) can further include (b) one or
more additional containers, vessels or compartments comprising: (i)
a first binding reagent comprising a linking agent, wherein the
first binding reagent is specific for a first analyte in the
sample, (ii) a first targeting agent complement comprising a
supplemental linking agent, provided in a separate container,
vessel or compartment or as a component of
[0092] (b)(i), (iii) a second binding reagent comprising a second
linking agent, wherein the second binding reagent is specific for a
second analyte in the sample, and (iv) a second targeting agent
complement comprising a second linking agent complement, provided
in a separate container, vessel or compartment or as a component of
(b)(iii).
[0093] Embodiment (21): a kit for measuring a plurality of
different analytes in a sample, the kit comprising: (a) a
container, vessel or compartment comprising on a solid support a
first targeting agent immobilized to a first region of the solid
support and a second targeting agent immobilized to a second region
of the solid support; and (b) one or more additional containers,
vessels or compartments comprising: (i) a first binding reagent
comprising a linking agent, wherein the first binding reagent is
specific for a first analyte in the sample, (ii) a first targeting
agent complement comprising a supplemental linking agent, provided
in a separate container, vessel or compartment or as a component of
(b)(i), (iii) a second binding reagent comprising a second linking
agent, wherein the second binding reagent is specific for a second
analyte in the sample, and (iv) a second targeting agent complement
comprising a second linking agent complement, provided in a
separate container, vessel or compartment or as a component of
(b)(iii).
[0094] Embodiment (22): a kit for measuring a plurality of
different analytes in a sample, the kit comprising: (a) a
container, vessel or compartment comprising on a solid support a
first targeting agent immobilized to a first region of the solid
support and a second targeting agent immobilized to a second region
of the solid support; and (b) four or more additional containers,
vessels or compartments comprising: (i) a first container
comprising a first binding reagent comprising a first linking
agent, wherein the first binding reagent is specific for a first
analyte in the sample, (ii) a second container comprising a first
targeting agent complement comprising a supplemental linking agent,
provided in a separate container, vessel or compartment, (iii) a
third container comprising a second binding reagent comprising a
second linking agent, wherein the second binding reagent is
specific for a second analyte in the sample, and (iv) a fourth
container comprising a second targeting agent complement comprising
a second linking agent complement, provided in a separate
container, vessel or compartment.
[0095] Embodiment (23): a kit for measuring a plurality of
different analytes in a sample, the kit comprising: (a) a
container, vessel or compartment comprising on a solid support a
first targeting agent immobilized to a first region of the solid
support and a second targeting agent immobilized to a second region
of the solid support; and (b) two or more additional containers,
vessels or compartments comprising: (i) a first container
comprising a first binding reagent comprising a first linking
agent, wherein the first binding reagent is specific for a first
analyte in the sample, and a first targeting agent complement
comprising a first linking agent complement, and (ii) a second
container comprising a second binding reagent comprising a second
linking agent, wherein the second binding reagent is specific for a
second analyte in the sample, and a second targeting agent
complement comprising a second linking agent complement.
[0096] Embodiment (24): a kit comprising (a) a multi-well plate
comprising a plurality of discrete binding domains each comprising
a first and second oligonucleotide, respectively, each of the first
and second oligonucleotides are selected from:
TABLE-US-00033 Sequence (5'-3') Acatcggtagtt (SEQ ID NO: 1)
Aactaccgatgt (SEQ ID NO: 2) acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0097] The kit of embodiment (24) can include one or more of the
following sequences:
TABLE-US-00034 Sequence acatcggtagtt (SEQ ID NO: 1) aactaccgatgt
(SEQ ID NO: 2) agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO:
6) atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
[0098] Embodiment (24) can further include instructions for use of
the multi-well plate in a method of conducting a binding assay for
a plurality of analytes, the method comprising the steps of: (a)
forming a first binding reagent complex comprising a first binding
reagent specific for a first analyte in the plurality of analytes
and the first oligonucleotide, wherein the first binding reagent is
bound to a linking agent and the first oligonucleotide is bound to
a supplemental linking agent wherein the first binding reagent
complex is formed by a reaction between the linking agent and the
supplemental linking agent; (b) forming a second binding reagent
complex comprising a second binding reagent specific for a second
analyte in the plurality of analytes and the second
oligonucleotide, wherein the second binding reagent is bound to a
second linking agent and the second oligonucleotide is bound to a
second linking agent complement wherein the second binding reagent
complex is formed by a reaction between the second linking agent
and the second linking agent complement; (c) mixing the first and
second binding reagent complexes with the two or more binding
domains each linked to a first oligonucleotide complement and a
second oligonucleotide complement, respectively, under conditions
sufficient to bind the first oligonucleotide to the first
oligonucleotide complement and the second oligonucleotide to the
second oligonucleotide complement; (d) mixing a sample comprising
the plurality of analytes to the mixture formed in step (c); (e)
adding a plurality of additional binding reagents to the mixture
formed in step (d), wherein the plurality of additional binding
reagents includes (i) a first detection reagent specific for the
first analyte and/or a first binding reagent-first analyte complex;
and (ii) a second detection reagent specific for the second analyte
and/or a second binding reagent-second analyte complex; and (f)
measuring the amount of the first and second analytes bound to the
binding domains.
[0099] In addition, embodiment (24) can include one or more of the
following elements: the first and second binding reagents each
comprise a receptor, ligand, antibody, hapten, antigen, epitope,
mimitope, aptamer, or an intercalater capable of binding to the
first and second analytes, respectively, e.g., the first and second
binding reagents each comprise an antibody capable of binding to
the first and second analyte, respectively; the first targeting
agent and the first targeting agent complement comprise an
oligonucleotide and a complementary oligonucleotide, a
receptor-ligand pair, an antigen-antibody pair, a hapten-antibody
pair, an antigen-antibody pair, an epitope-antibody pair, an
mimitope-antibody pair, an aptamer-target molecule pair,
hybridization partners, or an intercalater-target molecule pair;
the second targeting agent and the second targeting agent
complement comprise an oligonucleotide and a complementary
oligonucleotide, a receptor-ligand pair, an antigen-antibody pair,
a hapten-antibody pair, an antigen-antibody pair, an
epitope-antibody pair, an mimitope-antibody pair, an aptamer-target
molecule pair, hybridization partners, or an intercalater-target
molecule pair; the first targeting agent and the first targeting
agent complement comprise an oligonucleotide and a complementary
oligonucleotide; the second targeting agent and the second
targeting agent complement comprise an oligonucleotide and a
complementary oligonucleotide; the linking complex comprises a
linking agent and a supplemental linking agent connected thereto,
e.g., the linking complex is formed by a binding interaction
between (a) a thiol group and a maleimide or iodoacetamide; (b) an
aldehyde and a hydrazide; or (c) an alkyne and an azide; or the (a)
the linking agent is biotin and supplemental linking agent is
streptavidin or avidin; (b) the linking agent is streptavidin or
avidin and the supplemental linking agent is biotin; (c) the
linking agent is a peptide and the supplemental linking agent is an
anti-peptide antibody; or (d) the linking agent is an anti-peptide
antibody and the supplemental linking agent is a peptide.
[0100] In addition, embodiment (24) can further comprise, in a
separate vial, container, or compartment, a plurality of detection
reagents. For example, the plurality of detection reagents can
comprise a detectable label, e.g., a subset of the plurality of
detection reagents comprise a detectable label. In one example, the
detectable label is an electrochemiluminescent label. The two or
more binding domains can be positioned on an electrode. Optionally,
the two or more binding domains are located within one or more
wells of a multi-well plate.
[0101] In a specific example of embodiment (24), the first
targeting agent and the first targeting agent complement comprise a
pair of oligonucleotides, wherein the pair is selected from:
TABLE-US-00035 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0102] In embodiment (24), the second targeting agent and the
second targeting agent complement comprise a pair of
oligonucleotides, wherein the pair is selected from:
TABLE-US-00036 pair # Sequence (5'-3') 1 Acatcggtagtt (SEQ ID NO:
1) Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0103] In another specific example of embodiment (24), the first
targeting agent and the first targeting agent complement comprise a
pair of oligonucleotides, wherein the pair is selected from:
TABLE-US-00037 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0104] Embodiment (24) can also include one or more of the
following: the first targeting agent and the first targeting agent
complement comprise a first oligonucleotide and a first
complementary oligonucleotide, respectively, and the first
oligonucleotide and the first complementary oligonucleotide each
comprise approximately 10 to 50 bases; the first targeting agent
and the first targeting agent complement comprise a first
oligonucleotide and a first complementary oligonucleotide,
respectively, and the first oligonucleotide and the first
complementary oligonucleotide each comprise approximately 10 to 25
bases; the first binding reagent is an antibody comprising a biotin
molecule and the first targeting agent comprises a first
oligonucleotide including a streptavidin molecule and the linking
complex is formed by a reaction between the biotin and streptavidin
molecules; the second binding reagent is an antibody comprising an
additional biotin molecule and the second targeting agent comprises
a second oligonucleotide including an additional streptavidin
molecule and the linking complex is formed by a reaction between
the additional biotin and the additional streptavidin molecules;
the second targeting agent and the second targeting agent
complement comprise a second oligonucleotide and a second
complementary oligonucleotide, respectively, and the second
oligonucleotide and the second complementary oligonucleotide each
comprise approximately 10 to 50 bases; the second targeting agent
and the second targeting agent complement comprise a second
oligonucleotide and a second complementary oligonucleotide,
respectively, and the second oligonucleotide and the second
complementary oligonucleotide each comprise approximately 10 to 25
bases; and/or the second binding reagent is an antibody comprising
a biotin molecule and the second targeting agent comprises a second
oligonucleotide including a streptavidin molecule and the linking
complex is formed by a reaction between the biotin and streptavidin
molecules.
[0105] In any one or more of the preceding embodiments, the amount
of the first binding reagent on the second binding domain can be
<1% the amount of the first binding reagent on the first binding
domain.
[0106] Embodiment (25): a kit comprising: a multi-well plate having
one or more copies of an oligonucleotide array within at least one
well(s) of the plate, the array is positioned on a plurality of
binding domains, wherein at least 4 of the binding domains have
immobilized thereon a different oligonucleotide sequence selected
from:
TABLE-US-00038 Acatcggtagtt (SEQ ID NO: 1) Aactaccgatgt (SEQ ID NO:
2) acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4)
agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)
aggttcagtgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8)
atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10)
atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)
attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)
cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)
caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)
catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20)
cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)
cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)
cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)
cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28)
ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30)
gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)
gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)
gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)
ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38)
gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)
tacccggaataa (SEQ ID NO: 41) ttattccgggta (SEQ ID NO: 42)
tgcttgacttgg (SEQ ID NO: 43) ccaagtcaagca (SEQ ID NO: 44)
ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)
ttgtctagcggc (SEQ ID NO: 47) gccgctagacaa (SEQ ID NO: 48)
tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)
[0107] wherein each of the different oligonucleotide sequences is
modified with a linking agent.
[0108] Embodiment (26): a kit comprising: a multi-well plate having
one or more copies of an oligonucleotide array within at least one
well(s) of the plate, the array is positioned on a plurality of
binding domains, wherein at least 4 of the binding domains have
immobilized thereon a different oligonucleotide sequence selected
from:
TABLE-US-00039 Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0109] Embodiments (25) and (26) can include one or more of the
following elements: the linking agent comprises a biotin,
streptavidin, avidin, amino group, thiol group, aldehyde group,
hydrazide group, azide group, alkyne group, maleimide group or
iodoacetamide group; the linking agent is streptavidin; the kit
further includes, in one or more separate vials, containers, or
compartments, (a) a set of binding reagent pairs, wherein each
binding reagent of the set is specific for an analyte in a sample
comprising a plurality of analytes, wherein optionally, a binding
reagent pair within the set comprises a first binding reagent
comprising a supplemental linking agent, and still further
optionally the kit further includes, in one or more separate vials,
containers, or compartments, a supplemental linking agent. In one
example, the binding reagent is an antibody.
[0110] The kits of the preceding embodiments, e.g., embodiments
(25) and (26), can further include, in one or more separate vials,
containers, or compartments, a labeling kit, as well as one or more
additional reagents comprising: an assay buffer, diluent, read
buffer, or combinations thereof. The labeling kit can include, in
one or more separate vials, containers, or compartments,
SULFO-TAG.TM. NHS ester, LC-biotin NHS ester, a spin column, a
labeling buffer solution, ECL read buffer, assay and antibody
buffers, assay and antibody diluents, or combinations thereof.
[0111] In embodiments (25) and (26), the array comprises at least 7
binding domains, or at least 10 binding domains, or at least 16
binding domains, or at least 25 binding domains. Moreover, the
plate can include at least 24 wells, or at least 96 wells, or at
least 384 wells. The array can also include at least 10
oligonucleotides, or at least 16 oligonucleotides, or at least 25
oligonucleotides.
[0112] In a specific example of any one of the preceding
embodiments, the number of binding reagent pairs in the set is
equivalent to the number of binding domains in the array, or the
number of binding reagent pairs in the set is less than the number
of binding domains in the array.
BRIEF DESCRIPTION OF DRAWINGS
[0113] The accompanying drawings are provided to illustrate rather
than limit the scope of the invention.
[0114] FIGS. 1(a)-(d) illustrate an assay format comprising the
direct conjugation of binding reagents, A', B', and C', to a
plurality of binding domains, X, Y, and Z, respectively, via
reactions between targeting agents, A'', B'', and C'', and
targeting agent complements, A''', B''', and C''', respectively.
The targeting agents are attached to binding reagents, A', B', and
C', on a series of binding domains, X, Y, and Z, to form binding
reagent complexes, A.sub.RC, B.sub.RC, and C.sub.RC, respectively
(panel (b)), which react with analytes A, B, and C, respectively.
The presence of analytes A, B, and C on the solid support is
detected by the addition of labeled detection reagents, A*, B*, and
C*, which react with analytes, A, B, and C, respectively (panel
(c)). FIG. 1(d) is an enlarged view of the binding reagent
complexes formed on binding domains X, Y, and Z, binding reagent
complexes A.sub.RC, B.sub.RC, and C.sub.RC, respectively.
[0115] FIGS. 2(a)-(d) illustrates an assay format comprising the
direct conjugation of binding reagents, e.g., antibodies, A', B',
and C', to a plurality of binding domains, X, Y, and Z,
respectively, via reactions between oligonucleotide targeting
agents, A'', B'', and C'', and oligonucleotide targeting agent
complements, A''', B''', and C''', respectively. The
oligonucleotide targeting agents are attached to binding reagents,
A', B', and C', on a series of binding domains, X, Y, and Z, to
form binding reagent complexes, A.sub.RC, B.sub.RC, and C.sub.RC,
respectively (panel (b)), which react with analytes A, B, and C,
respectively. The presence of analytes A, B, and C on the solid
support is detected by the addition of labeled detection reagents,
A*, B*, and C*, which react with analytes, A, B, and C,
respectively (panel (c)). FIG. 2(d) is an enlarged view of the
binding reagent complexes formed on binding domains X, Y, and Z,
binding reagent complexes A.sub.RC, B.sub.RC, and C.sub.RC,
respectively.
[0116] FIGS. 3(a)-(e) illustrates an assay format involving the
conjugation of a plurality of binding reagents, A', B', and C', to
a plurality of binding domains, X, Y, and Z, respectively, via a
series of linking complexes. As shown in panel (a), binding
reagents A', B', and C' are attached to linking agents, L.sub.A,
L.sub.B, and L.sub.C, while targeting agents A'', B'', and C' are
attached to supplemental linking agents, L.sub.A', L.sub.B', and
L.sub.C', respectively. Binding reagents A', B', and C' are mixed
with targeting agents to form binding reagent complexes, A.sub.RC,
B.sub.RC, and C.sub.RC, respectively. The binding reagent complexes
formed in panel (a) are mixed with a plurality of binding domains,
X, Y, and Z, to which targeting agent complements, A''', B''', and
C''' are bound (panel (b)) to adhere the binding reagent complexes
to the binding domains (see panel (c)). A sample comprising
analytes A, B, and C, is added to the mixture, and simultaneously
or sequentially, a set of detection reagents, A*, B*, and C* are
also added to detect analytes bound to the binding domains. FIG.
3(e) is an enlarged view of the binding reagent complexes formed on
binding domains X, Y, and Z, binding reagent complexes A.sub.RC,
B.sub.RC, and C.sub.RC, respectively.
[0117] FIGS. 4(a)-(e) illustrates an assay format involving the
conjugation of a plurality of binding reagents, A', B', and C', to
a plurality of binding domains, X, Y, and Z, respectively, via a
series of linking complexes. As shown in panel (a), binding
reagents A', B', and C' are attached to linking agents, L.sub.A,
L.sub.B, and L.sub.C, while oligonucleotide targeting agents A'',
B'', and C' are attached to supplemental linking agents, L.sub.A',
L.sub.B', and L.sub.C', respectively. Binding reagents A', B', and
C' are mixed with oligonucleotide targeting agents to form binding
reagent complexes, A.sub.RC, B.sub.RC, and C.sub.RC, respectively.
The binding reagent complexes formed in panel (a) are mixed with a
plurality of binding domains, X, Y, and Z, to which oligonucleotide
targeting agent complements, A''', B''', and C''' are bound (panel
(b)) to adhere the binding reagent complexes to the binding domains
(see panel (c)). A sample comprising analytes A, B, and C, is added
to the mixture, and simultaneously or sequentially, a set of
detection reagents, A*, B*, and C* are also added to detect
analytes bound to the binding domains. FIG. 4(e) is an enlarged
view of the binding reagent complexes formed on binding domains X,
Y, and Z, binding reagent complexes A.sub.RC, B.sub.RC, and
C.sub.RC, respectively.
[0118] FIGS. 5(a)-(d) illustrate various combinations of reagents
that can be used in the assay format of the invention. In FIG.
5(a), all reagents are mixed with the sample in a single step,
whereas in FIG. 5(b), all reagents are mixed and then added to the
surface, and in 5(c), modified binding reagents and modified
targeting agent complements are mixed, added to a surface bearing a
plurality of binding domains each including a targeting agent, and
the surface-mixture is mixed with the sample and detection reagents
in one step or two (one step addition of sample and detection
reagents is shown in FIG. 5(c) and the sequential addition of
sample and detection reagents is shown in FIG. 5(d).
[0119] FIGS. 6(a)-(f) show various modified surfaces, e.g.,
particles and substrates bearing a plurality of binding domains
that can be used in the assay format of the invention.
[0120] FIG. 7 shows a procedure to chemically modify an antibody
with sulfo-SMCC to yield a maleimide-activated antibody which can
be conjugated to an oligonucleotide via a thiol group on the
oligonucleotide.
[0121] FIGS. 8(a)-(g) show the results of a direct assay format on
a 7-plex chemokine panel and a 10-plex TH1/TH2 panel.
[0122] FIGS. 9(a)-(c) show a procedure for the production and use
of a multi-well assay plate in an indirect assay format using
biotinylated capture antibodies with oligonucleotide modified with
streptavidin molecules.
[0123] FIGS. 10(a)-(c) show a procedure for the production and use
of a multi-well assay plate in an indirect assay format using
biotinylated capture antibodies, neat streptavidin, and
biotinylated oligonucleotides.
[0124] FIGS. 11(a)-(g) show a comparison of LOD values for three
different oligonucleotide-mediated assay formats for a cytokine B
panel.
DETAILED DESCRIPTION OF THE INVENTION
[0125] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. The articles "a" and "an" are used herein to refer to one
or to more than one (i.e., to at least one) of the grammatical
object of the article. By way of example, "an element" means one
element or more than one element.
[0126] The present invention provides flexible solid phase binding
assay formats that allow a user or manufacturer to configure an
assay based on specific user requirements. The methods and kits
described herein provide a flexible platform for creating a
multiplexed binding assay for a plurality of target analytes. With
a support including a plurality of binding domains bearing a series
of generic targeting agent complements, it is possible to configure
a multiplexed assay for any set of analytes. One only needs to
select which analytes will be evaluated in which binding domain and
pair the appropriate binding reagents and targeting agents with
each selected binding domain. Using this platform, a user can build
a personalized assay panel.
[0127] Such flexible multiplexed assay formats can be achieved
using the methods and products disclosed herein. For example, a
method of conducting a multiplexed binding assay for a plurality of
analytes of interest can be implemented using the following
steps:
[0128] (a) combining, in one or more steps, the following
components: [0129] (i) a sample comprising a first analyte of
interest and a second analyte of interest, [0130] (ii) a first
targeting agent immobilized on a first binding domain, [0131] (iii)
a first targeting agent complement connected to a linking agent,
wherein the first targeting agent complement is a binding partner
of the first targeting agent, [0132] (iv) a first binding reagent
connected to a supplemental linking agent, wherein the first
binding reagent is a binding partner of the first analyte, [0133]
(v) a second targeting agent immobilized on a second binding
domain, [0134] (vi) a second targeting agent complement connected
to a linking agent, wherein the second targeting agent complement
is a binding partner of the second targeting agent, [0135] (vii) a
second binding reagent connected to a supplemental linking agent,
wherein the second binding reagent is a binding partner of the
second analyte, and [0136] (viii) optionally, at least two copies
of a bridging agent, [0137] wherein, if the bridging agent is
omitted, each linking agent is a binding partner of the
supplemental linking agent, or if the bridging agent is included,
the bridging agent has a first binding site for one of the linking
agents and an additional binding site for one of the supplemental
linking agents;
[0138] (b) forming [0139] (i) a first binding complex on the first
binding domain comprising the first targeting agent, the first
targeting agent complement, the first binding reagent and the first
analyte, and [0140] (ii) a second binding complex on the second
binding domain comprising the second targeting agent, the second
targeting agent complement, the second binding reagent and the
second analyte, and
[0141] (c) measuring the amount of the first and second analytes on
the first and second binding domains, respectively.
[0142] In one embodiment, if a bridging agent is not used, the
method includes (a) combining components (i)-(vii) in one or more
steps, (b) forming the first and second binding complexes on the
first and second binding domains, respectively, and (c) measuring
the amount of the first and second analytes on the first and second
binding domains, respectively. For example, the first targeting
agent complement and the first binding reagent can be provided as a
pre-bound first targeting complex including the first targeting
agent complement and the first binding reagent linked through a
binding interaction between the linking agent and supplemental
linking agent; and likewise, the second targeting agent complement
and the second binding reagent can be provided as a pre-bound
second targeting complex comprising the second targeting agent
complement and the second binding reagent linked through a binding
interaction between the linking agent and supplemental linking
agent. In this embodiment, the first targeting complex can be
provided pre-bound to the first targeting agent immobilized on the
first binding domain; and likewise, the second targeting complex
can be provided pre-bound to the second targeting agent immobilized
on the second binding domain. When the first and second binding
reagents are provided in pre-bound targeting complexes, the
combining step may further includes combining the first and second
targeting complexes with the sample to form a mixture thereof,
binding the first analyte to the first binding reagent in the first
targeting complex and binding the second analyte to the second
binding reagent in the second targeting complex, contacting a
mixture of the first and second targeting complexes bound to first
and second analytes, respectively, with the first and second
binding domains. The binding complexes on the first and second
domains are thereby formed by binding the first targeting complex
to the first targeting agent on the first binding domain and
binding the second targeting complex to the second targeting agent
on the second binding domain. Moreover, the combining step can
further include combining the first and second targeting complexes
with the sample; and binding the first analyte to the first binding
reagent in the first targeting complex and binding the second
analyte to the second binding reagent in the second targeting
complex.
[0143] In another alternative embodiment, the combining step (a)
includes the steps of combining, in a first volume of liquid, said
first targeting agent complement, said first binding reagent and,
if used, said bridging reagent and linking said first targeting
agent complement and said first binding reagent through their
attached linking agents to form a first targeting complex; and
combining, in a second volume of liquid, said second targeting
agent complement, said second binding reagent and, if used, said
bridging reagent and linking said second targeting agent complex
complement and said second binding reagent through their attached
linking agents to form a second targeting complex. In this
embodiment, the combining step (a) can also include the steps of
combining said first and second targeting complexes, contacting the
combination of said first and second targeting complexes with said
first and second binding domains, and binding said first targeting
complex to said first targeting agent on said first binding domain
and binding said second targeting complex to said second targeting
agent on said second binding domain. In this embodiment, the
combining step further includes combining the combination of the
first and second targeting complexes with the sample and binding
the first analyte to the first binding reagent in the first
targeting complex and binding the second analyte to the second
binding reagent in the second targeting complex. The first and
second targeting complexes can be combined with the sample prior to
contacting the first and second targeting complexes with the first
and second binding domains; the first and second targeting
complexes can be combined with the sample after contacting the
first and second targeting complexes with the first and second
binding domains; or the first and second targeting complexes can be
combined with the sample and contacted with the first and second
binding domains at the same time.
[0144] If a bridging agent is included in the method, then the
linking agent and supplemental linking agents each bind to the
bridging agent, and the combining step therefore brings those
elements attached to the linking agents and supplemental linking
agents together. For example, the combining step (a) includes
combining, in a first volume of liquid, (xi) the first targeting
agent complement, the first binding reagent and the bridging
reagent and further includes forming the first targeting complex by
linking the first targeting agent complement and the first binding
reagent through a bridging complex including the linking agent
bound to the bridging agent to which the supplemental linking agent
is bound. Combining step (a) also includes combining, in a second
volume of liquid, (xii) the second targeting agent complement, the
second binding reagent and the bridging reagent and further
includes forming the second targeting complex by linking the second
targeting agent complement and the second binding reagent through a
bridging complex including the linking agent bound to the bridging
agent to which the supplemental linking agent is bound. In this
embodiment, the combining step (a) can also include combining
(xiii) the first and second targeting complexes, and combining the
combination of the first and second targeting complexes with the
first and second binding domains, and binding the first targeting
complex on the first binding domain and binding the second
targeting complex on the second binding domain. In this embodiment,
the combining step further includes combining (xiv) the first and
second targeting complexes with the sample and binding the first
analyte to the first binding reagent in the first targeting complex
and binding the second analyte to the second binding reagent in the
second targeting complex The first and second targeting complexes
can be combined with the sample prior to contacting the first and
second targeting complexes with the first and second binding
domains; the first and second targeting complexes can be combined
with the sample after contacting the first and second targeting
complexes with the first and second binding domains; or the first
and second targeting complexes can be combined with the sample and
contacted with the first and second binding domains at the same
time.
[0145] The methods described herein can be used to multiplex a
plurality of analytes of interest in a sample. In this regard, the
sample contains one or more additional analytes of interest and for
each additional analyte of interest, the combining step (a) further
comprises combining, in one or more steps, (ix) an additional
targeting agent immobilized on an additional binding domain, an
additional targeting agent complement connected to a linking agent,
and an additional binding reagent connected to a supplemental
linking agent, and (x) an additional binding complex on the
additional binding domain comprising the additional targeting
agent, the additional targeting agent complement, the additional
binding reagent and the additional analyte; the forming step (b)
further comprises forming (iii) an additional binding complex on
the additional binding domain comprising the additional targeting
agent, the additional targeting agent complement, the additional
binding reagent and the additional analyte; and the measurement in
step (c) further comprises measuring the amount of the additional
analyte on the additional binding domain.
[0146] In a specific embodiment, the invention includes a method of
conducting a binding assay for a plurality of analytes comprising
(a) contacting a sample with two or more binding domains linked to
at least a first and second binding reagent that each bind a first
and second analyte, respectively, of the plurality of analytes to
form complexes comprising the first analyte bound to the first
binding reagent and the second analyte bound to the second binding
reagent, wherein (x) the first binding domain comprises a first
binding reagent complex comprising (i) a first targeting agent
bound to the first binding domain and to a first targeting agent
complement; and (ii) the first binding reagent bound to the first
targeting agent complement via a linking complex; and (y) the
second binding domain comprises a second binding reagent complex
comprising (i) a second targeting agent bound to the second binding
domain and to a second targeting agent complement; and (ii) the
second binding reagent bound to the second targeting agent
complement via a linking complex; (b) contacting the first and
second binding reagent complexes with a plurality of detection
reagents comprising a first detection reagent that binds the first
analyte or a complex comprising the first analyte, and a second
detection reagent that binds the second analyte or a complex
comprising the second analyte; and (c) measuring the amount of the
first and second analytes bound to the two or more binding
domains.
[0147] In another specific embodiment, the method includes: (a)
forming a first binding reagent complex comprising a first binding
reagent specific for a first analyte in the plurality of analytes
and a first targeting agent, wherein the first binding reagent is
bound to a linking agent and the first targeting agent is bound to
a supplemental linking agent wherein the first binding reagent
complex is formed by a reaction between the linking agent and the
supplemental linking agent; (b) forming a second binding reagent
complex comprising a second binding reagent specific for a second
analyte in the plurality of analytes and a second targeting agent,
wherein the second binding reagent is bound to a second linking
agent and the second targeting agent is bound to a second linking
agent complement wherein the second binding reagent complex is
formed by a reaction between the second linking agent and the
second linking agent complement; (c) mixing the first and second
binding reagent complexes with two or more binding domains each
linked to a first targeting agent complement and a second targeting
agent complement, respectively, under conditions sufficient to bind
the first targeting agent to the first targeting agent complement
and the second targeting agent to the second targeting agent
complement; (d) mixing a sample comprising the plurality of
analytes to the mixture formed in step (c); (e) adding a plurality
of additional binding reagents to the mixture formed in step (d),
wherein the plurality of additional binding reagents includes (i) a
first detection reagent specific for the first analyte and/or a
first binding reagent-first analyte complex; and (ii) a second
detection reagent specific for the second analyte and/or a second
binding reagent-second analyte complex; and (f) measuring the
amount of the first and second analytes bound to the binding
domains.
[0148] A further specific embodiment includes (a) forming a first
binding reagent complex comprising a first binding reagent specific
for a first analyte in the plurality of analytes and a first
targeting agent, wherein the first binding reagent is bound to a
linking agent and the first targeting agent is bound to a
supplemental linking agent wherein the first binding reagent
complex is formed by a reaction between the linking agent and the
supplemental linking agent; (b) forming a second binding reagent
complex comprising a second binding reagent specific for a second
analyte in the plurality of analytes and a second targeting agent,
wherein the second binding reagent is bound to a second linking
agent and the second targeting agent is bound to a second linking
agent complement wherein the second binding reagent complex is
formed by a reaction between the second linking agent and the
second linking agent complement; (c) mixing the first and second
binding reagent complexes and the sample with two or more binding
domains each linked to a first targeting agent complement and a
second targeting agent complement, respectively, under conditions
sufficient to bind the first targeting agent to the first targeting
agent complement and the second targeting agent to the second
targeting agent complement; (d) adding a plurality of additional
binding reagents to the mixture formed in step (c), wherein the
plurality of additional binding reagents includes (i) a first
detection reagent specific for the first analyte and/or a first
binding reagent-first analyte complex; and (ii) a second detection
reagent specific for the second analyte and/or a second binding
reagent-second analyte complex; and (e) measuring the amount of the
first and second analytes bound to the binding domains.
[0149] Specific embodiments of the method of the present invention
are illustrated in FIGS. 3-6. FIGS. 1 and 2 illustrate a direct
assay method that does not involve a linking complex. These figures
are provided for comparative purposes. FIG. 1 illustrates a direct
multiplexed assay for analytes A, B, and C. Binding reagents
specific for these analytes, A', B', and C', respectively, are
attached to targeting agents, A'', B'', and C''. A solution
including these binding reagents attached to their corresponding
targeting agents is mixed with a solid phase to which targeting
agent complements A''', B''', and C''', respectively are bound to a
series of discrete binding domains. The binding reagents are
adsorbed to the surface to form binding reagent complexes,
A.sub.RC, B.sub.RC, and C.sub.RC, each binding reagent affixed via
the targeting agent complements to a discrete binding domain on the
surface. The surface is contacted with a sample comprising analytes
A, B, and C, as well as detection binding reagents, A*, B*, and C*,
which are capable of binding to analytes A, B, and C, respectively,
and/or a complex comprising those analytes. The detection binding
reagents include a detectable label. Alternatively, the surface is
contacted with a sample comprising the plurality of analytes and
subsequently contacted with a mixture of detection binding
reagents. Once the detection binding reagents are bound to the
surface, and optionally, the surface is washed to remove unbound
reagents, the presence of each analyte is detected via the
detection reagents bound to each discrete binding domain. FIG. 2
illustrates a specific embodiment of FIG. 1 involving the use of
antibodies as binding reagents and oligonucleotide-complementary
oligonucleotide pairs as targeting agent/targeting agent complement
pairs. It will be evident to the skilled artisan that the direct
methods illustrated in FIGS. 1 and 2 are not configurable by the
user. Each individual binding domain includes a predetermined
targeting agent complement, such that only a single binding
reagent-targeting agent can bind to a single binding domain in the
array.
[0150] FIGS. 3-4 illustrate particular embodiments of the instant
invention that offer the user optimal flexibility in a user-defined
assay configuration. FIG. 3 illustrates an indirect binding format
for analytes A, B, and C, incorporating a series of linking
complexes that allow the user to tailor the assay for his/her
needs. FIG. 3(a)-(b) illustrates a general approach for making the
targeting complexes of the invention: a series of solutions are
formed that include one of the binding reagents (A', B', and C')
bound to a linking agents (L.sub.A, L.sub.B, and L.sub.C,
respectively). The solutions also include the corresponding
targeting agents, (A'' for A', B'' for B', and C'' for C'), bound
to a supplemental linking agent (L.sub.A', L.sub.B', and L.sub.C',
respectively). The solutions are mixed to form the mixture of
binding reagent-linking complex-targeting agent complexes shown in
panel (b). An advantage of this approach is that it does not
require the linking reagents for each targeting complex to be
non-cross reactive and, in fact, allows the linking agents to be
used in each targeting complex (i.e., L.sub.A=L.sub.B=L.sub.C and
L.sub.A'=L.sub.B'=L.sub.C'). In one embodiment illustrated in FIG.
3(c)-(e) the mixture of binding reagent-linking complex-targeting
agent complexes are mixed with a surface comprising a plurality of
discrete binding domains to which targeting agent complements,
A''', B''', and C''' are bound. The binding reagent-linking
complex-targeting agent complexes are adsorbed to form binding
reagent complexes, A.sub.RC, B.sub.RC, and C.sub.RC as shown in
panel (c). An expanded view of the binding reagent complexes is
shown in FIG. 3(e). The surface is contacted with a sample
comprising analytes A, B, and C, as well as detection binding
reagents, A*, B*, and C*, which are capable of binding to analytes
A, B, and C, respectively, and/or a complex comprising those
analytes. The detection binding reagents include a detectable
label. Alternatively, the surface is contacted with a sample
comprising the plurality of analytes and subsequently contacted
with a mixture of detection binding reagents. Once the detection
binding reagents are bound to the surface, and optionally, the
surface is washed to remove unbound reagents, the presence of each
analyte is detected via the detection reagents bound to each
discrete binding domain (panel 3(d)). FIG. 4 illustrates a specific
embodiment of FIG. 3 involving the use of antibodies as binding
reagents and oligonucleotide-complementary oligonucleotide pairs as
targeting agent/targeting agent complement pairs. As noted for FIG.
3, the linking agents for each binding reagent may be the same and
the linking agent complements for each targeting agent may be the
same.
[0151] The skilled artisan will readily appreciate that various
permutations of the assay format depicted in FIGS. 3-4 are
possible. Certain preferred embodiments are depicted in FIG.
5(a)-(c). For example, all of the reagents, i.e., binding reagents
modified by supplemental linking agents, targeting agent
complements modified by linking agents, detection reagents and
sample, can be mixed together with the surface bearing targeting
agent-modified binding domains in a single step to form the
complexes shown in FIGS. 3(d) and 4(d), optionally washed, and
analyzed for the presence of analytes A, B, and C, bound to the
surface (FIG. 5(a)). Alternatively, binding reagents modified by
supplemental linking agents, and targeting agents modified by
linking agents can be mixed in a single step, added to the surface
having targeting agent-modified binding domains in a subsequent
step, sample and detection reagents are added, and analyzed in a
final step (FIG. 5(b)). In yet another embodiment, binding reagents
modified by supplemental linking agents, and targeting agent
complements modified by linking agents can be mixed, added to the
surface bearing targeting agents in discrete binding domains, mixed
with sample, and then detection reagents are added (FIG. 5(c)).
Individual analyte solutions can be added to each binding domain
sequentially or simultaneously in a single mixture, and likewise,
individual detection reagents can be added to each binding domain
sequentially or simultaneously in a single mixture. Any surface
binding step can optionally be followed by a washing step to remove
any unbound components of the assay before proceeding to the next
step.
[0152] The invention also provides kits, components, and
consumables that can be used to practice the methods described
herein. The following materials/methods are used in the instant
invention.
[0153] (i) Samples/Analytes
[0154] Examples of samples that may be analyzed by the methods of
the present invention include, but are not limited to food samples
(including food extracts, food homogenates, beverages, etc.),
environmental samples (e.g., soil samples, environmental sludges,
collected environmental aerosols, environmental wipes, water
filtrates, etc.), industrial samples (e.g., starting materials,
products or intermediates from an industrial production process),
human clinical samples, veterinary samples and other samples of
biological origin. Biological samples that may be analyzed include,
but are not limited to, feces, mucosal swabs, physiological fluids
and/or samples containing suspensions of cells. Specific examples
of biological samples include blood, serum, plasma, feces, mucosal
swabs, tissue aspirates, tissue homogenates, cell cultures and cell
culture supernatants (including cultures of eukaryotic and
prokaryotic cells), urine, saliva, sputum, and cerebrospinal
fluid.
[0155] Analytes that may be measured using the methods of the
invention include, but are not limited to proteins, toxins, nucleic
acids, microorganisms, viruses, cells, fungi, spores,
carbohydrates, lipids, glycoproteins, lipoproteins,
polysaccharides, drugs, hormones, steroids, nutrients, metabolites
and any modified derivative of the above molecules, or any complex
comprising one or more of the above molecules or combinations
thereof. The level of an analyte of interest in a sample may be
indicative of a disease or disease condition or it may simply
indicate whether the patient was exposed to that analyte.
[0156] The assays of the present invention may be used to determine
the concentration of one or more, e.g., two or more analytes in a
sample. Thus, two or more analytes may be measured in the same
sample. Panels of analytes that can be measured in the same sample
include, for example, panels of assays for analytes or activities
associated with a disease state or physiological conditions.
Certain such panels include panels of cytokines and/or their
receptors (e.g., one or more of TNF-alpha, TNF-beta, IL1-alpha,
IL1-beta, IL2, IL4, IL6, IL-10, IL-12, IFN-y, etc.), growth factors
and/or their receptors (e.g., one or more of EGF, VGF, TGF, VEGF,
etc.), drugs of abuse, therapeutic drugs, vitamins, pathogen
specific antibodies, auto-antibodies (e.g., one or more antibodies
directed against the Sm, RNP, SS-A, SS-alpha, J0-1, and Scl-70
antigens), allergen-specific antibodies, tumor markers (e.g., one
or more of CEA, PSA, CA-125 II, CA 15-3, CA 19-9, CA 72-4, CYFRA
21-1, NSE, AFP, etc.), markers of cardiac disease including
congestive heart disease and/or acute myocardial infarction (e.g.,
one or more of Troponin T, Troponin I, myoglobin, CKMB,
myeloperoxidase, glutathione peroxidase, .beta.-natriuretic protein
(BNP), alpha-natriuretic protein (ANP), endothelin, aldosterone,
C-reactive protein (CRP), etc.), markers associated with hemostasis
(e.g., one or more of Fibrin monomer, D-dimer,
thrombin-antithrombin complex, prothrombin fragments 1 & 2,
anti-Factor Xa, etc.), markers of acute viral hepatitis infection
(e.g., one or more of IgM antibody to hepatitis A virus, IgM
antibody to hepatitis B core antigen, hepatitis B surface antigen,
antibody to hepatitis C virus, etc.), markers of Alzheimers Disease
(alpha-amyloid, beta-amyloid, A.beta. 42, A.beta. 40, A.beta. 38,
A.beta. 39, A.beta. 37, A.beta. 34, tau-protein, etc.), markers of
osteoporosis (e.g., one or more of cross-linked Nor C-telopeptides,
total deoxypyridinoline, free deoxypyridinoline, osteocalcin,
alkaline phosphatase, C-terminal propeptide of type I collagen,
bone-specific alkaline phosphatase, etc.), markers of fertility
state or fertility associated disorders (e.g., one or more of
Estradiol, progesterone, follicle stimulating hormone (FSH),
lutenizing hormone (LH), prolactin, hCG, testosterone, etc.),
markers of thyroid disorders (e.g., one or more of thyroid
stimulating hormone (TSH), Total T3, Free T3, Total T4, Free T4,
and reverse T3), and markers of prostrate cancer (e.g., one or more
of total PSA, free PSA, complexed PSA, prostatic acid phosphatase,
creatine kinase, etc.). Certain embodiments of invention include
measuring, e.g., one or more, two or more, four or more or 10 or
more analytes associated with a specific disease state or
physiological condition (e.g., analytes grouped together in a
panel, such as those listed above; e.g., a panel useful for the
diagnosis of thyroid disorders may include e.g., one or more of
thyroid stimulating hormone (TSH), Total T3, Free T3, Total T4,
Free T4, and reverse T3).
[0157] The methods of the present invention are designed to allow
detection of a wide variety of biological and biochemical agents,
as described above. In one embodiment, the methods may be used to
detect pathogenic and/or potentially pathogenic virus, bacteria and
toxins including biological warfare agents ("BWAs") in a variety of
relevant clinical and environmental matrices, including and without
limitation, blood, sputum, stool, filters, swabs, etc. A
non-limiting list of pathogens and toxins that may be analyzed
(alone or in combination) using the methods of the present
invention is Bacillus anthracia (anthrax), Yersinia pestis
(plague), Vibrio cholerae (cholera), Francisella tularensis
(tularemia), Brucella spp. (Brucellosis), Coxiella burnetii (Q
fever), listeria, salmonella, shigella, V. cholera, Chlamydia
trachomatis, Burkholderia pseudomallei, orthopox viruses including
variola virus (smallpox), viral encephalitis, Venezuelan equine
encephalitis virus (VEE), western equine encephalitis virus (WEE),
eastern equine encephalitis virus (EEE), Alphavirus, viral
hemorrhagic fevers, Arenaviridae, Bunyaviridae, Filoviridae,
Flaviviridae, Ebola virus, staphylococcal enterotoxins, ricin,
botulinum toxins (A, B, E), Clostridium botulinum, mycotoxin,
Fusarium, Myrotecium, Cephalosporium, Trichoderma,
Verticimonosporium, Stachybotrys, glanders, wheat fungus, Bacillus
globigii, Serratia marcescens, yellow rain, trichothecene
mycotoxins, Salmonella typhimurium, aflatoxin, Xenopsylla cheopis,
Diamanus montanus, alastrim, monkeypox, Arenavirus, Hantavirus,
Lassa fever, Argentine hemorrhagic fevers, Bolivian hemorrhagic
fevers, Rift Valley fever virus, Crimean-Congo virus, Hanta virus,
Marburg hemorrhagic fevers, yellow fever virus, dengue fever
viruses, influenza (including human and animal strains including
H5N1 avian influenza, influenza A, influenza A, H1 specific,
influenza A, H3 specific, influenza A, H5 specific, influenza A,
2009-H1N1 specific, influenza B), RSV, human immunodeficiency
viruses I and II (HIV I and II), hepatitis A, hepatitis B,
hepatitis C, hepatitis (non-A, B or C), Enterovirus, Epstein-Barr
virus, Cytomegalovirus, herpes simplex viruses, Chlamydia
trachomatis, Neisseria gonorrheae, Trichomonas vaginalis, human
papilloma virus, Treponema pallidum, Streptococcus pneumonia,
Borellia burgdorferi, Haemophilus influenzae, Mycoplasma
pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila,
Staphylococcus aureus, Staphylococcus Enterotoxin B (SEB), Abrin,
Shiga Toxin 1, Shiga Toxin 2, Moraxella catarrhalis, Streptococcus
pyogenes, Clostridium difficile, Neisseria meningitidis, Klebsiella
pneumoniae, Mycobacterium tuberculosis, Group A streptococcus, E.
Coli O157, coronavirus, Coxsackie A virus, rhinovirus,
parainfluenza virus, respiratory syncytial virus (RSV),
metapneumovirus, vaccinia, and adenovirus.
[0158] (ii) Binding Reagents
[0159] The skilled artisan in the field of binding assays will
readily appreciate the scope of binding agents and companion
binding partners that may be used in the present methods. A
non-limiting list of such pairs include (in either order)
oligonucleotides and complements, receptor/ligand pairs,
antibodies/antigens, natural or synthetic receptor/ligand pairs,
amines and carbonyl compounds (i.e., binding through the formation
of a Schiff's base), hapten/antibody pairs, antigen/antibody pairs,
epitope/antibody pairs, mimitope/antibody pairs, aptamer/target
molecule pairs, hybridization partners, and intercalater/target
molecule pairs.
[0160] In a preferred embodiment, the binding assays of the methods
of the present invention employ antibodies or other receptor
proteins as binding reagents. The term "antibody" includes intact
antibody molecules (including hybrid antibodies assembled by in
vitro re-association of antibody subunits), antibody fragments and
recombinant protein constructs comprising an antigen binding domain
of an antibody (as described, e.g., in Porter, R. R. and Weir, R.
C. J. Cell Physiol., 67 (Suppl); 51-64 (1966) and Hochman, 1.
Inbar, D. and Givol, D. Biochemistry 12: 1130 (1973)), as well as
antibody constructs that have been chemically modified, e.g., by
the introduction of a detectable label.
[0161] iii) Targeting Agents, Linking Agents & Bridging
Agents
[0162] Binding reagents are linked to components that enable their
attachment to each other and/or to solid phases, directly or
indirectly. These components are referred to herein as targeting
agents and linking agents. As used herein, targeting agents and
their complements are used to adhere a binding reagent to a surface
or support, whereas linking agents and supplemental linking agents
are used to attach a binding reagent to a targeting agent, directly
or indirectly through a bridging agent, if one is used.
[0163] In one embodiment, a targeting agent and its complement
comprise a first oligonucleotide and a complementary
oligonucleotide, a receptor-ligand pair, an antigen-antibody pair,
a hapten-antibody pair, an epitope-antibody pair, a
mimetope-antibody pair, an aptamer-target molecule pair,
hybridization partners, or an intercalator-target molecule pair.
The targeting agents and complements used in an assay are selected
such that the targeting agents and complements associated with a
binding reagent for an analyte (for example, a first analyte)
measured by the assay are substantially non-cross-reactive with the
targeting agents and complements associated with the binding
reagents for the other analytes measured by the assay (for example,
a second analyte). Accordingly, in an assay of the invention, the
binding of a binding reagent to its associated binding domain
(through its associated targeting agent and targeting agent
complement) should be substantially greater than its binding to
binding domains associated with other analytes (and presenting
different targeting agent complements). Preferably the
cross-reactivity for the binding of binding reagents for an analyte
to binding domains associated with other analytes relative to the
binding to the correct binding domain is <1%, more preferably
<0.1% and more preferably <0.01%. In a preferred embodiment,
the targeting agent/targeting agent complement comprise a pair of
oligonucleotides including complementary sequences and the
targeting agent and its complement are contacted under conditions
sufficient to hybridize the targeting agent to its complement.
[0164] The preferred length is approximately 5 to 100 bases,
preferably, approximately, 10 to 50 bases, and more preferably
approximately 10 to 25 bases. In addition, the targeting
oligonucleotides sequences need not be identical in length and in
certain embodiments it may be beneficial to provide one targeting
oligonucleotide sequence that is longer than its binding partner,
e.g., by up to 25 bases, or up to 15 bases, or up to 10 bases.
Oligonucleotide sequences and their complements can be generated by
techniques known in the art for generating pairs of complementary
oligonucleotides with similar binding energies (or melting
temperatures) and low inter-pair cross-reactivity (e.g., commercial
or public software for selecting probes or primers for multiplexed
nucleic acid assays). Oligonucleotide sequences can include
naturally occurring nucleic acid bases as well as non-naturally
occurring and/or modified bases. For example, the oligonucleotide
sequences can include Iso-dC and/or Iso-dG, which are chemical
variants of cytosine and guanine, respectively, available from
EraGen Biosciences, Inc. (www.eragen.com). Incorporation of such
modified bases into oligonucleotide sequences effectively expands
the genetic alphabet and permits synthesis of oligonucleotides that
have increased specificity and decreased mismatch hybridization
potential. For example, an oligonucleotide containing Iso-dC can be
designed so that it will hybridize to a complementary oligo
containing Iso-dG but will not hybridize to any naturally occurring
nucleic acids sequence. In addition or alternatively,
oligonucleotide sequences and their complements can include
dimerized and/or dendritic oligonucleotide sequences. Moreover, the
oligonucleotide sequences and their complements can be
multi-functional, e.g., including (a) a first segment designed to
bind to a capture reagent via a first segment complement (i.e., the
capture reagent includes a targeting agent complement that is
complementary to the first segment of the multi-functional
targeting agent), and (b) a second segment designed to bind to an
additional moiety present in the assay medium. In this regard,
reference is made to copending application serial no.
PCT/US15/30925, filed May 15, 2015.
[0165] In one approach, a computer algorithm can be used to
generate oligonucleotide sequence pairs based on one or more, and
preferably all, of the following rules: (i) GC content between
about 40-60%, e.g., 40-50%; (ii) a maximum string of base repeats
in a sequence of no more than three; (iii) a maximum number of base
pair matches of six between sequences in different pairs, with no
more than four matches in a row; (iv) a rejection of sequences with
predicted hairpin loop sizes between 2-5, oligonucleotides if they
have four or more base-pair matches in the stem region (loop sizes
of six or greater are retained); and (v) a higher free energy
(.DELTA.G.degree.) of the specific interactions resulting from a
40-60% or 40-50% GC content (.DELTA.G.degree. is dependent on
temperature and salt concentration, and at 23.degree. C. and 200 mM
of a monovalent cation, pH 7.0, .DELTA.G.degree. preferably exceeds
-15 kcal/mol). In a particular embodiment, at least relative GC
content and .DELTA.G.degree. are considered, along with one or more
the rules identified above, in oligonucleotide selection. In one
embodiment, it may be advantageous to design oligonucleotide
sequences that minimize non-specific binding and this can be
achieved by a variety of methods. For example, one can design
oligonucleotide/oligonucleotide pairs that form no more than three
consecutive G/C pairs with other oligonucleotides used in the
assay. Alternatively or additionally, one or more of the following
configurations can be avoided: formation of single nucleotide loops
or single nucleotide mismatches positioned between G/C-rich
sequences when paired with other oligonucleotides used in the
assay.
[0166] In one embodiment, the targeting agent and the targeting
agent complement comprise a pair of oligonucleotides, wherein the
pair is selected from one of the following sequence pairs in Table
1(a):
TABLE-US-00040 pair # Sequence (5'-3') 1 Acatcggtagtt (SEQ ID NO:
1) Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)
gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)
ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)
ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)
gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50)
[0167] In a particular embodiment, the targeting agent and the
targeting agent complement comprise a pair of oligonucleotides,
wherein the pair is selected from one of the following sequence
pairs in Table 1(b):
TABLE-US-00041 Pair Name Modification Sequence 1 3' Thiol Oligo
12b-1 3'-thiol C3 SS acatcggtagtt (SEQ ID NO: 1) 3' Biotin Oligo
12b-1 3' biotin aactaccgatgt (SEQ ID NO: 2) 3 3' Thiol Oligo 12b-5
3'-thiol C3 SS agaagaagatcc (SEQ ID NO: 5) 3' Biotin Oligo 12b-5 3'
biotin ggatcttcttct (SEQ ID NO: 6) 6 3' Thiol Oligo 12b-12 3'-thiol
C3 SS atcattaccacc (SEQ ID NO: 11) 3' Biotin Oligo 12b-12 3' biotin
ggtggtaatgat (SEQ ID NO: 12) 7 3' Thiol Oligo 12b-14 3'-thiol C3 SS
attaacgggagc (SEQ ID NO: 13) 3' Biotin Oligo 12b-14 3' biotin
gctcccgttaat (SEQ ID NO: 14) 8 3' Thiol Oligo 12b-17 3'-thiol C3 SS
cagaggtcttaa (SEQ ID NO: 15) 3' Biotin Oligo 12b-17 3' biotin
ttaagacctctg (SEQ ID NO: 16) 9 3' Thiol Oligo 12b-18 3'-thiol C3 SS
caggtgtccatt (SEQ ID NO: 17) 3' Biotin Oligo 12b-18 3' biotin
aatggacacctg (SEQ ID NO: 18) 11 3' Thiol Oligo 12b-20 3'-thiol C3
SS cctacgatatac (SEQ ID NO: 21) 3' Biotin Oligo 12b-20 3' biotin
gtatatcgtagg (SEQ ID NO: 22) 12 3' Thiol Oligo 12b-21 3'-thiol C3
SS cgaatgtagagt (SEQ ID NO: 23) 3' Biotin Oligo 12b-21 3' biotin
actctacattcg (SEQ ID NO: 24) 13 3' Thiol Oligo 12b-22 3'-thiol C3
SS cggtttgagata (SEQ ID NO: 25) 3' Biotin Oligo 12b-22 3' biotin
tatctcaaaccg (SEQ ID NO: 26) 16 3' Thiol Oligo 12b-26 3'-thiol C3
SS gacataaagcga (SEQ ID NO: 31) 3' Biotin Oligo 12b-26 3' biotin
tcgctttatgtc (SEQ ID NO: 32) 17 3' Thiol Oligo 12b-28 3'-thiol C3
SS gccatagtctct (SEQ ID NO: 33) 3' Biotin Oligo 12b-28 3' biotin
agagactatggc (SEQ ID NO: 34) 18 3' Thiol Oligo 12b-30 3'-thiol C3
SS gctaattcacca (SEQ ID NO: 35) 3' Biotin Oligo 12b-30 3' biotin
tggtgaattagc (SEQ ID NO: 36) 20 3' Thiol Oligo 12b-33 3'-thiol C3
SS gttgattctgtc (SEQ ID NO: 39) 3' Biotin Oligo 12b-33 3' biotin
gacagaatcaac (SEQ ID NO: 40) 23 3' Thiol Oligo 12b-41 3'-thiol C3
SS ttccacttaggg (SEQ ID NO: 45) 3' Biotin Oligo 12b-41 3' biotin
ccctaagtggaa (SEQ ID NO: 46) 25 3' Thiol Oligo 12b-43 3'-thiol C3
SS tttcccttgcta (SEQ ID NO: 49) 3' Biotin Oligo 12b-43 3' biotin
tagcaagggaaa (SEQ ID NO: 50)
[0168] For example, the invention includes one of the sets of ten
pair of targeting agent and the targeting agent complement shown in
Table 1(c):
TABLE-US-00042 Set (1) 3' Thiol Oligo Corresponding 3' Biotinylated
Oligo 5'-ATC ATT ACC ACC/3ThioMC3-D/-3' 5'-GGT GGT AAT GAT/3Bio/-3'
(SEQ ID NO: 11) (SEQ ID NO: 12) 5'-CCT ACG ATA TAC/3ThioMC3-D/-3'
5'-GTA TAT CGT AGG/3Bio/-3' (SEQ ID NO: 21) (SEQ ID NO: 22) 5'-CGG
TTT GAG ATA/3ThioMC3-D/-3' 5'-TAT CTC AAA CCG/3Bio/-3' (SEQ ID NO:
25) (SEQ ID NO: 26) 5'-GAC ATA AAG CGA/3ThioMC3-D/-3' 5'-TCG CTT
TAT GTC/3Bio/-3' (SEQ ID NO: 31) (SEQ ID NO: 32) 5'-GTT GAT TCT
GTC/3ThioMC3-D/-3' 5'-GAC AGA ATC AAC/3Bio/-3' (SEQ ID NO: 39) (SEQ
ID NO: 40) 5'-GCT AAT TCA CCA/3ThioMC3-D/-3' 5'-TGG TGA ATT
AGC/3Bio/-3' (SEQ ID NO: 35) (SEQ ID NO: 36) 5'-TTT CCC TTG
CTA/3ThioMC3-D/-3' 5'-TAG CAA GGG AAA/3Bio/-3' (SEQ ID NO: 49) (SEQ
ID NO: 50) 5'-AGA AGA AGA TCC/3ThioMC3-D/-3' 5'-GGA TCT TCT
TCT/3Bio/-3' (SEQ ID NO: 5) (SEQ ID NO: 6) 5'-ACA TCG GTA
GTT/3ThioMC3-D/-3' 5'-AAC TAC CGA TGT/3Bio/-3' (SEQ ID NO: 1) (SEQ
ID NO: 2 5'-GCC ATA GTC TCT/3ThioMC3-D/-3' 5'-AGA GAC TAT
GGC/3Bio/-3' (SEQ ID NO: 33) (SEQ ID NO: 34) Set (2) 5'-CGA ATG TAG
AGT/3ThioMC3-D/-3' 5'-ACT CTA CAT TCG/3Bio/-3' (SEQ ID NO: 23) (SEQ
ID NO: 24) 5'-TTC CAC TTA GGG/3ThioMC3-D/-3' 5'-CCC TAA GTG
GAA/3Bio/-3' (SEQ ID NO: 45) (SEQ ID NO: 46) 5'-CAG AGG TCT
TAA/3ThioMC3-D/-3' 5'-TTA AGA CCT CTG/3Bio/-3' (SEQ ID NO: 15) (SEQ
ID NO: 16) 5'-ACG TCC CAG TTG/3ThioMC3-D/-3' 5'-CAA CTG GGA
CGT/3Bio/-3' (SEQ ID NO: 3) (SEQ ID NO: 4) 5'-AGG TTC AGT
GCA/3ThioMC3-D/-3' 5'-TGC ACT GAA CCT/3Bio/-3' (SEQ ID NO: 7) (SEQ
ID NO: 8) 5'-ATC AGG ATA CGC/3ThioMC3-D/-3' 5'-GCG TAT CCT
GAT/3Bio/-3' (SEQ ID NO: 9) (SEQ ID NO: 10) 5'-CAT CCA ATC
CAG/3ThioMC3-D/-3' 5'-CTG GAT TGG ATG/3Bio/-3' (SEQ ID NO: 19) (SEQ
ID NO: 20) 5'-CTT ACA ACG CCA/3ThioMC3-D/-3' 5'-TGG CGT TGT
AAG/3Bio/-3' (SEQ ID NO: 27) (SEQ ID NO: 28) 5'-CTT TCT CGG
CAC/3ThioMC3-D/-3' 5'-GTG CCG AGA AAG/3Bio/-3' (SEQ ID NO: 29) (SEQ
ID NO: 30) 5'-GGT CGT GTT TCA/3ThioMC3-D/-3' 5'-TGA AAC ACG
ACC/3Bio/-3' (SEQ ID NO: 37) (SEQ ID NO: 38) Set (3) 5'-ATT AAC GGG
AGC/3ThioMC3-D/-3' 5'-GCT CCC GTT AAT/3Bio/-3' (SEQ ID NO: 13) (SEQ
ID NO: 14) 5'-CAG GTG TCC ATT/3ThioMC3-D/-3' 5'-AAT GGA CAC
CTG/3Bio/-3' (SEQ ID NO: 17) (SEQ ID NO: 18) 5'-CGA ATG TAG
AGT/3ThioMC3-D/-3' 5'-ACT CTA CAT TCG/3Bio/-3' (SEQ ID NO: 23) (SEQ
ID NO: 24) 5'-TTC CAC TTA GGG/3ThioMC3-D/-3' 5'-CCC TAA GTG
GAA/3Bio/-3' (SEQ ID NO: 45) (SEQ ID NO: 46) 5'-CAG AGG TCT
TAA/3ThioMC3-D/-3' 5'-TTA AGA CCT CTG/3Bio/-3' (SEQ ID NO: 15) (SEQ
ID NO: 16) 5'-ACG TCC CAG TTG/3ThioMC3-D/-3' 5'-CAA CTG GGA
CGT/3Bio/-3' (SEQ ID NO: 3) (SEQ ID NO: 4) 5'-AGG TTC AGT
GCA/3ThioMC3-D/-3' 5'-TGC ACT GAA CCT/3Bio/-3' (SEQ ID NO: 7) (SEQ
ID NO: 8) 5'-ATC AGG ATA CGC/3ThioMC3-D/-3' 5'-GCG TAT CCT
GAT/3Bio/-3' (SEQ ID NO: 9) (SEQ ID NO: 10) 5'-CAT CCA ATC
CAG/3ThioMC3-D/-3' 5'-CTG GAT TGG ATG/3Bio/-3' (SEQ ID NO: 19) (SEQ
ID NO: 20) 5'-CTT ACA ACG CCA/3ThioMC3-D/-3' 5'-TGG CGT TGT
AAG/3Bio/-3' (SEQ ID NO: 27) (SEQ ID NO: 28) Set (4) 5'-GCT AAT TCA
CCA/3ThioMC3-D/-3' 5'-TGG TGA ATT AGC/3Bio/-3' (SEQ ID NO: 35) (SEQ
ID NO: 36) 5'-TTT CCC TTG CTA/3ThioMC3-D/-3' 5'-TAG CAA GGG
AAA/3Bio/-3' (SEQ ID NO: 49) (SEQ ID NO: 50) 5'-AGA AGA AGA
TCC/3ThioMC3-D/-3' 5'-GGA TCT TCT TCT/3Bio/-3' (SEQ ID NO: 5) (SEQ
ID NO: 6) 5'-ACA TCG GTA GTT/3ThioMC3-D/-3' 5'-AAC TAC CGA
TGT/3Bio/-3' (SEQ ID NO: 1) (SEQ ID NO: 2) 5'-GCC ATA GTC
TCT/3ThioMC3-D/-3' 5'-AGA GAC TAT GGC/3Bio/-3' (SEQ ID NO: 33) (SEQ
ID NO: 34) 5'-ATT AAC GGG AGC/3ThioMC3-D/-3' 5'-GCT CCC GTT
AAT/3Bio/-3' (SEQ ID NO: 13) (SEQ ID NO: 14) 5'-CAG GTG TCC
ATT/3ThioMC3-D/-3' 5'-AAT GGA CAC CTG/3Bio/-3' (SEQ ID NO: 17) (SEQ
ID NO: 18) 5'-CGA ATG TAG AGT/3ThioMC3-D/-3' 5'-ACT CTA CAT
TCG/3Bio/-3' (SEQ ID NO: 23) (SEQ ID NO: 24) 5'-TTC CAC TTA
GGG/3ThioMC3-D/-3' 5'-CCC TAA GTG GAA/3Bio/-3' (SEQ ID NO: 45) (SEQ
ID NO: 46) 5'-CAG AGG TCT TAA/3ThioMC3-D/-3' 5'-TTA AGA CCT
CTG/3Bio/-3' (SEQ ID NO: 15) (SEQ ID NO: 16) Set (5) 5'-ATC ATT ACC
ACC/3ThioMC3-D/-3' 5'-GGT GGT AAT GAT/3Bio/-3' (SEQ ID NO: 11) (SEQ
ID NO: 12) 5'-CCT ACG ATA TAC/3ThioMC3-D/-3' 5'-GTA TAT CGT
AGG/3Bio/-3' (SEQ ID NO: 21) (SEQ ID NO: 22) 5'-CGG TTT GAG
ATA/3ThioMC3-D/-3' 5'-TAT CTC AAA CCG/3Bio/-3' (SEQ ID NO: 25) (SEQ
ID NO: 26) 5'-GAC ATA AAG CGA/3ThioMC3-D/-3' 5'-TCG CTT TAT
GTC/3Bio/-3' (SEQ ID NO: 31) (SEQ ID NO: 32) 5'-GTT GAT TCT
GTC/3ThioMC3-D/-3' 5'-GAC AGA ATC AAC/3Bio/-3' (SEQ ID NO: 39) (SEQ
ID NO: 40) 5'-ATC AGG ATA CGC/3ThioMC3-D/-3' 5'-GCG TAT CCT
GAT/3Bio/-3' (SEQ ID NO: 9) (SEQ ID NO: 10) 5'-CAT CCA ATC
CAG/3ThioMC3-D/-3' 5'-CTG GAT TGG ATG/3Bio/-3' (SEQ ID NO: 19) (SEQ
ID NO: 20) 5'-CTT ACA ACG CCA/3ThioMC3-D/-3' 5'-TGG CGT TGT
AAG/3Bio/-3' (SEQ ID NO: 27) (SEQ ID NO: 28) 5'-CTT TCT CGG
CAC/3ThioMC3-D/-3' 5'-GTG CCG AGA AAG/3Bio/-3' (SEQ ID NO: 29) (SEQ
ID NO: 30) 5'-GGT CGT GTT TCA/3ThioMC3-D/-3' 5'-TGA AAC ACG
ACC/3Bio/-3' (SEQ ID NO: 37) (SEQ ID NO: 38) Set (6) 5'-ATC ATT ACC
ACC/3ThioMC3-D/-3' 5'-GGT GGT AAT GAT/3Bio/-3' (SEQ ID NO: 11) (SEQ
ID NO: 12) 5'-CCT ACG ATA TAC/3ThioMC3-D/-3' 5'-GTA TAT CGT
AGG/3Bio/-3' (SEQ ID NO: 21) (SEQ ID NO: 22) 5'-GAC ATA AAG
CGA/3ThioMC3-D/-3' 5'-TCG CTT TAT GTC/3Bio/-3' (SEQ ID NO: 31) (SEQ
ID NO: 32) 5'-GTT GAT TCT GTC/3ThioMC3-D/-3' 5'-GAC AGA ATC
AAC/3Bio/-3' (SEQ ID NO: 39) (SEQ ID NO: 40) 5'-AGA AGA AGA
TCC/3ThioMC3-D/-3' 5'-GGA TCT TCT TCT/3Bio/-3' (SEQ ID NO: 5) (SEQ
ID NO: 6) 5'-ACA TCG GTA GTT/3ThioMC3-D/-3' 5'-AAC TAC CGA
TGT/3Bio/-3' (SEQ ID NO: 1) (SEQ ID NO: 2 5'-CAG GTG TCC
ATT/3ThioMC3-D/-3' 5'-AAT GGA CAC CTG/3Bio/-3' (SEQ ID NO: 17) (SEQ
ID NO: 18) 5'-CGA ATG TAG AGT/3ThioMC3-D/-3' 5'-ACT CTA CAT
TCG/3Bio/-3' (SEQ ID NO: 23) (SEQ ID NO: 24) 5'-ACG TCC CAG
TTG/3ThioMC3-D/-3' 5'-CAA CTG GGA CGT/3Bio/-3' (SEQ ID NO: 3) (SEQ
ID NO: 4) 5'-AGG TTC AGT GCA/3ThioMC3-D/-3' 5'-TGC ACT GAA
CCT/3Bio/-3' (SEQ ID NO: 7) (SEQ ID NO: 8) Set (7) 5'-CGG TTT GAG
ATA/3ThioMC3-D/-3' 5'-TAT CTC AAA CCG/3Bio/-3' (SEQ ID NO: 25) (SEQ
ID NO: 26) 5'-GAC ATA AAG CGA/3ThioMC3-D/-3' 5'-TCG CTT TAT
GTC/3Bio/-3' (SEQ ID NO: 31) (SEQ ID NO: 32) 5'-TTT CCC TTG
CTA/3ThioMC3-D/-3' 5'-TAG CAA GGG AAA/3Bio/-3' (SEQ ID NO: 49) (SEQ
ID NO: 50) 5'-AGA AGA AGA TCC/3ThioMC3-D/-3' 5'-GGA TCT TCT
TCT/3Bio/-3' (SEQ ID NO: 5) (SEQ ID NO: 6) 5'-ATT AAC GGG
AGC/3ThioMC3-D/-3' 5'-GCT CCC GTT AAT/3Bio/-3' (SEQ ID NO: 13) (SEQ
ID NO: 14) 5'-CAG GTG TCC ATT/3ThioMC3-D/-3' 5'-AAT GGA CAC
CTG/3Bio/-3' (SEQ ID NO: 17) (SEQ ID NO: 18) 5'-TTC CAC TTA
GGG/3ThioMC3-D/-3' 5'-CCC TAA GTG GAA/3Bio/-3' (SEQ ID NO: 45) (SEQ
ID NO: 46) 5'-CAG AGG TCT TAA/3ThioMC3-D/-3' 5'-TTA AGA CCT
CTG/3Bio/-3' (SEQ ID NO: 15) (SEQ ID NO: 16) 5'-ATC AGG ATA
CGC/3ThioMC3-D/-3' 5'-GCG TAT CCT GAT/3Bio/-3' (SEQ ID NO: 9) (SEQ
ID NO: 10) 5'-CAT CCA ATC CAG/3ThioMC3-D/-3' 5'-CTG GAT TGG
ATG/3Bio/-3' (SEQ ID NO: 19) (SEQ ID NO: 20) Set (8) 5'-GCT AAT TCA
CCA/3ThioMC3-D/-3' 5'-TGG TGA ATT AGC/3Bio/-3' (SEQ ID NO: 35) (SEQ
ID NO: 36) 5'-TTT CCC TTG CTA/3ThioMC3-D/-3' 5'-TAG CAA GGG
AAA/3Bio/-3' (SEQ ID NO: 49) (SEQ ID NO: 50) 5'-AGA AGA AGA
TCC/3ThioMC3-D/-3' 5'-GGA TCT TCT TCT/3Bio/-3' (SEQ ID NO: 5) (SEQ
ID NO: 6) 5'-ACA TCG GTA GTT/3ThioMC3-D/-3' 5'-AAC TAC CGA
TGT/3Bio/-3' (SEQ ID NO: 1) (SEQ ID NO: 2 5'-GCC ATA GTC
TCT/3ThioMC3-D/-3' 5'-AGA GAC TAT GGC/3Bio/-3' (SEQ ID NO: 33) (SEQ
ID NO: 34) 5'-ACG TCC CAG TTG/3ThioMC3-D/-3' 5'-CAA CTG GGA
CGT/3Bio/-3' (SEQ ID NO: 3) (SEQ ID NO: 4) 5'-AGG TTC AGT
GCA/3ThioMC3-D/-3' 5'-TGC ACT GAA CCT/3Bio/-3' (SEQ ID NO: 7) (SEQ
ID NO: 8) 5'-ATC AGG ATA CGC/3ThioMC3-D/-3' 5'-GCG TAT CCT
GAT/3Bio/-3'
(SEQ ID NO: 9) (SEQ ID NO: 10) 5'-CAT CCA ATC CAG/3ThioMC3-D/-3'
5'-CTG GAT TGG ATG/3Bio/-3' (SEQ ID NO: 19) (SEQ ID NO: 20) 5'-CTT
ACA ACG CCA/3ThioMC3-D/-3' 5'-TGG CGT TGT AAG/3Bio/-3' (SEQ ID NO:
27) (SEQ ID NO: 28) Set (9) 5'-ATT AAC GGG AGC/3ThioMC3-D/-3'
5'-GCT CCC GTT AAT/3Bio/-3' (SEQ ID NO: 13) (SEQ ID NO: 14) 5'-CAG
GTG TCC ATT/3ThioMC3-D/-3' 5'-AAT GGA CAC CTG/3Bio/-3' (SEQ ID NO:
17) (SEQ ID NO: 18) 5'-CGA ATG TAG AGT/3ThioMC3-D/-3' 5'-ACT CTA
CAT TCG/3Bio/-3' (SEQ ID NO: 23) (SEQ ID NO: 24) 5'-TTC CAC TTA
GGG/3ThioMC3-D/-3' 5'-CCC TAA GTG GAA/3Bio/-3' (SEQ ID NO: 45) (SEQ
ID NO: 46) 5'-CAG AGG TCT TAA/3ThioMC3-D/-3' 5'-TTA AGA CCT
CTG/3Bio/-3' (SEQ ID NO: 15) (SEQ ID NO: 16) 5'-ATC AGG ATA
CGC/3ThioMC3-D/-3' 5'-GCG TAT CCT GAT/3Bio/-3' (SEQ ID NO: 9) (SEQ
ID NO: 10) 5'-CAT CCA ATC CAG/3ThioMC3-D/-3' 5'-CTG GAT TGG
ATG/3Bio/-3' (SEQ ID NO: 19) (SEQ ID NO: 20) 5'-CTT ACA ACG
CCA/3ThioMC3-D/-3' 5'-TGG CGT TGT AAG/3Bio/-3' (SEQ ID NO: 27) (SEQ
ID NO: 28) 5'-CTT TCT CGG CAC/3ThioMC3-D/-3' 5'-GTG CCG AGA
AAG/3Bio/-3' (SEQ ID NO: 29) (SEQ ID NO: 30) 5'-GGT CGT GTT
TCA/3ThioMC3-D/-3' 5'-TGA AAC ACG ACC/3Bio/-3' (SEQ ID NO: 37) (SEQ
ID NO: 38) Set (10) 5'-GAC ATA AAG CGA/3ThioMC3-D/-3' 5'-TCG CTT
TAT GTC/3Bio/-3' (SEQ ID NO: 31) (SEQ ID NO: 32) 5'-GTT GAT TCT
GTC/3ThioMC3-D/-3' 5'-GAC AGA ATC AAC/3Bio/-3' (SEQ ID NO: 39) (SEQ
ID NO: 40) 5'-GCT AAT TCA CCA/3ThioMC3-D/-3' 5'-TGG TGA ATT
AGC/3Bio/-3' (SEQ ID NO: 35) (SEQ ID NO: 36) 5'-TTT CCC TTG
CTA/3ThioMC3-D/-3' 5'-TAG CAA GGG AAA/3Bio/-3' (SEQ ID NO: 49) (SEQ
ID NO: 50) 5'-AGA AGA AGA TCC/3ThioMC3-D/-3' 5'-GGA TCT TCT
TCT/3Bio/-3' (SEQ ID NO: 5) (SEQ ID NO: 6) 5'-TTC CAC TTA
GGG/3ThioMC3-D/-3' 5'-CCC TAA GTG GAA/3Bio/-3' (SEQ ID NO: 45) (SEQ
ID NO: 46) 5'-CAG AGG TCT TAA/3ThioMC3-D/-3' 5'-TTA AGA CCT
CTG/3Bio/-3' (SEQ ID NO: 15) (SEQ ID NO: 16) 5'-ACG TCC CAG
TTG/3ThioMC3-D/-3' 5'-CAA CTG GGA CGT/3Bio/-3' (SEQ ID NO: 3) (SEQ
ID NO: 4) 5'-AGG TTC AGT GCA/3ThioMC3-D/-3' 5'-TGC ACT GAA
CCT/3Bio/-3' (SEQ ID NO: 7) (SEQ ID NO: 8) 5'-ATC AGG ATA
CGC/3ThioMC3-D/-3' 5'-GCG TAT CCT GAT/3Bio/-3' (SEQ ID NO: 9) (SEQ
ID NO: 10)
[0169] In a specific embodiment, the set comprises set 1 listed in
Table 1(c). Alternatively, the set comprises set 2 from Table 1(c);
the set can also comprise set 3 from Table 1(c); the set further
comprises set 4 from Table 1(c); the set also includes set 5 from
Table 1(c); the set includes set 6 from Table 1(c); the set further
comprises set 7 from Table 1(c); the set can also include set 8
from Table 1(c); the set includes set 9 from Table 1(c); and/or the
set includes set 10 from Table 1(c).
[0170] The targeting agent and targeting agent complement may be
present in a 1:1 ratio. Alternatively, the targeting agent may be
present in an excess, e.g., in a 2:1 ratio of targeting agent to
targeting agent complement, to increase the likelihood of binding
the targeting agent to its complement.
[0171] In one embodiment, suitable linking agents and supplemental
linking agents include chemical moieties that react to form a
linking complex. For example, the linking complex is formed by a
binding interaction between chemical moieties present on the
linking agent and supplemental linking agent, e.g., a thiol group
and a maleimide or iodoacetamide groups; an aldehyde and a
hydrazide; or an alkyne and an azide.
[0172] Alternatively, a linking complex can be formed by a
protein-protein binding reaction between a linking agent and a
supplemental linking agent. For example, a protein-protein binding
reaction can be formed via binding between a receptor/ligand pair,
hapten/antibody pair, antigen/antibody pair, epitope/antibody pair,
mimitope/antibody pair, aptamer/target molecule pair, hybridization
partners, and intercalater/target molecule pair. In one embodiment,
the linking agent is biotin and the supplemental linking agent is
streptavidin or avidin (or vice versa); or the linking agent is a
peptide and the supplemental linking agent is an anti-peptide
antibody (or vice versa).
[0173] Certain embodiments described herein employ linking agents
that bind directly to supplemental linking agents. In these and
other such embodiments, the linking and supplemental linking agents
that bind to each other can be replaced with linking and
supplemental linking agents that can concurrently bind to a
bridging agent. In these alternate embodiments, a bridging agent is
included in the mixture of the linking agent and supplemental
linking agent, when forming the targeting complex. A bridging
agent, if one is used, binds to the linking agent and the
supplemental linking agent. For example, the bridging agent
includes a binding site for the linking agent and an additional
binding site for the supplemental linking agent, and the
combination of the three components, i.e., the bridging agent, the
linking agent, and the supplemental linking agent, comprises a
bridging complex. In one embodiment, the bridging agent is
streptavidin or avidin (each of which are tetramers with four
independent binding sites for biotin) and the linking agent and
supplemental linking agent are each biotin, such that the bridging
complex comprises (biotin-(streptavidin (or avidin))-biotin).
[0174] In a preferred embodiment, a linking complex is formed by a
binding reaction between a linking agent, L.sub.A, and a
supplemental linking agent, L.sub.A'. A multiplex assay format may
be configured to detect analytes A, B, and C, and therefore, the
reagent complexes designed to interact with those individual
analytes may include linking agents and supplemental linking agents
selected from L.sub.A and L.sub.A', L.sub.B and L.sub.B', and
L.sub.C and L.sub.C'. Each of the linking agent/supplemental
linking agent pairs used to construct the binding complexes may be
the same or different. In a preferred embodiment, each of the
linking agent/supplemental linking agent pairs comprise the same
set of reagents. In a particularly preferred embodiment, each of
the linking agent/supplemental linking agent pairs comprise, e.g.,
a biotin molecule as the linking agent on a binding reagent and a
streptavidin or avidin molecule on a targeting agent as the
supplemental linking agent (or vice versa). In addition, a linking
complex can also be formed by a binding reaction between a biotin
molecule on a binding reagent and a streptavidin or avidin molecule
on a targeting agent, wherein the streptavidin or avidin molecule
is bound to the targeting agent via a reaction with a biotin
molecule (acting as a bridging agent) on the targeting agent. In a
preferred embodiment, once a linking complex is formed between a
biotin and streptavidin molecule on a binding reagent and a
targeting agent, respectively (or vice versa), excess free biotin
molecule can be added to prevent cross-reactivity between
additional binding reagents and targeting agents that may be
combined in solution.
[0175] (iv) Solid Phases
[0176] A wide variety of solid phases are suitable for use in the
methods of the present invention including conventional solid
phases from the art of binding assays. Solid phases may be made
from a variety of different materials including polymers (e.g.,
polystyrene and polypropylene), ceramics, glass, composite
materials (e.g., carbon-polymer composites such as carbon-based
inks) Suitable solid phases include the surfaces of macroscopic
objects such as an interior surface of an assay container (e.g.,
test tubes, cuvettes, flow cells, cartridges, wells in a multi-well
plate, etc.), slides, assay chips (such as those used in gene or
protein chip measurements), pins or probes, beads, filtration
media, lateral flow media (for example, filtration membranes used
in lateral flow test strips), etc.
[0177] Suitable solid phases also include particles (including but
not limited to colloids or beads) commonly used in other types of
particle-based assays e.g., magnetic, polypropylene, and latex
particles, materials typically used in solid-phase synthesis e.g.,
polystyrene and polyacrylamide particles, and materials typically
used in chromatographic applications e.g., silica, alumina,
polyacrylamide, polystyrene. The materials may also be a fiber such
as a carbon fibril. Microparticles may be inanimate or
alternatively, may include animate biological entities such as
cells, viruses, bacterium and the like. A particle used in the
present method may be comprised of any material suitable for
attachment to one or more binding reagents, and that may be
collected via, e.g., centrifugation, gravity, filtration or
magnetic collection. A wide variety of different types of particles
that may be attached to binding reagents are sold commercially for
use in binding assays. These include non-magnetic particles as well
as particles comprising magnetizable materials which allow the
particles to be collected with a magnetic field. In one embodiment,
the particles are comprised of a conductive and/or semiconductive
material, e.g., colloidal gold particles. The microparticles may
have a wide variety of sizes and shapes. By way of example and not
limitation, microparticles may be between 5 nanometers and 100
micrometers. Preferably microparticles have sizes between 20 nm and
10 micrometers. The particles may be spherical, oblong, rod-like,
etc., or they may be irregular in shape.
[0178] FIG. 6(a)-(c) illustrates various set of particles that can
be used in the present invention. FIG. 6(a) shows a set of
particles, provided in one or more vials, containers, or
compartments, that are each modified with a distinct targeting
agent. Alternatively, as shown in FIG. 6(b), a set of particles can
be provided in one or more vials, containers, or compartments, that
are each modified with a distinct binding reagent, the binding
reagent being attached to the particle in a targeting complex that
comprises the binding reagent (through linking a supplemental
linking agents) to a targeting agent complement that is bound to a
targeting agent on the surface of the particle. Still further, FIG.
6(c) shows yet another embodiment in which a mixed set of particles
is provided, wherein a subset includes particles that are each
modified with a distinct binding reagent (as in FIG. 6(b) and a
subset includes particles that are each modified with a distinct
targeting agent (as in FIG. 6(a)). The particles shown in FIG. 6(c)
include a subset of preconfigured particles with binding reagents
for a pre-determined set of analytes and a subset of non-configured
particles that can be modified by a user to attach an additional
set of binding reagents for an additional set of analytes, i.e., by
binding the particles to an additional set of targeting complexes
comprising additional binding reagents. These additional reagents
may be provided by the user thereby allowing the user to add a set
of user-defined assays to a pre-defined set of assays.
[0179] The particles used in the present method may be coded to
allow for the identification of specific particles or
subpopulations of particles in a mixture of particles. The use of
such coded particles has been used to enable multiplexing of assays
employing particles as solid phase supports for binding assays. In
one approach, particles are manufactured to include one or more
fluorescent dyes and specific populations of particles are
identified based on the intensity and/or relative intensity of
fluorescence emissions at one or more wave lengths. This approach
has been used in the Luminex xMAP systems (see, e.g., U.S. Pat. No.
6,939,720) and the Becton Dickinson Cytometric Bead Array systems.
Alternatively, particles may be coded through differences in other
physical properties such as size, shape, imbedded optical patterns
and the like. As indicated by the cross-hatching of the particles
in FIGS. 6(a)-(c), one or more particles provided in a mixture or
set of particles may be coded to be distinguishable from other
particles in the mixture by virtue of particle optical properties,
size, shape, imbedded optical patterns and the like.
[0180] Alternatively or additionally, the binding reagents can be
bound via binding reagent complexes to different discrete binding
domains on one or more solid phases, e.g., as in a binding array,
such that discrete assay signals are generated on each binding
domain and therefore, the different analytes bound to those domains
can be measured independently. In one example of such an
embodiment, the binding domains are prepared by immobilizing, on
one or more surfaces, discrete domains of targeting agents that,
through a binding reagent complex built on the individual domains,
are configured to bind analytes of interest. Optionally, the
surface(s) may define, in part, one or more boundaries of a
container (e.g., a flow cell, well, cuvette, etc.) which holds the
sample or through which the sample is passed. In a preferred
embodiment, individual binding domains are formed on electrodes for
use in electrochemical or electrochemiluminescence assays.
Multiplexed measurement of analytes on a surface comprising a
plurality of binding domains using electrochemiluminescence has
been used in the Meso Scale Diagnostics, LLC, MULTI-ARRAY.RTM. and
SECTOR.RTM. Imager line or products (see, e.g., U.S. Pat. Nos.
7,842,246 and 6,977,722, the disclosures of which are incorporated
herein by reference in their entireties).
[0181] FIG. 6(d)-(f) illustrate various alternative plate formats
including distinct binding domains that can be used in the present
invention. FIG. 6(d) shows a surface bearing a plurality of binding
domains that are each modified with a distinct targeting agent.
Alternatively, as shown in FIG. 6(e), a surface can be provided
bearing a plurality of binding domains that are each modified with
a distinct binding reagent, the binding reagent being attached to
the binding domain in a targeting complex that comprises the
binding reagent (through linking a supplemental linking agents) to
a targeting agent complement that is bound to a targeting agent on
the binding domain. Still further, FIG. 6(f) shows yet another
embodiment in which a mixed set of binding domains on a surface is
provided, wherein a subset includes binding domains that are each
modified with a distinct binding reagent (as in FIG. 6e) and a
subset includes binding domains that are each modified by a
distinct targeting agent (as in FIG. 6(d)). The binding domains
shown in FIG. 6(f) include a subset of preconfigured binding
domains with binding reagents for a pre-determined set of analytes
and a subset of non-configured binding domains that can be modified
by a user to attach an additional set of binding reagents for an
additional set of analytes, i.e., by binding these non-configured
binding domains to an additional set of targeting complexes
comprising additional binding reagents. These additional reagents
may be provided by the user, thereby allowing the user to add a set
of user-defined assays to a pre-defined set of assays.
[0182] Reagents, i.e., targeting agents, can be bound to a surface
by known methods, e.g., established methods for modifying particles
or for forming arrays. One non-limiting example of a method of
attaching a protein or oligonucleotide to a surface is illustrated
in FIG. 7. This method uses
Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(Sulfo-SMCC), a well-established heterobifunctional cross-linking
agent. Reaction of the N-hydroxysuccinimide (NHS) group of SMCC
with bovine serum albumin (BSA) labels the BSA with thiol-reactive
maleimide groups. The maleimide groups are, in turn, reacted with
thiol-modified oligonucleotides to form BSA-oligonucleotide
conjugates that are linked through stable thioether bonds. Arrays
of these reagents can be formed by printing patterns of the
reagents on surfaces that adsorb or react with proteins (such as
BSA), thereby generating patterned arrays of the associated
oligonucleotides. In one specific example, arrays are formed by
printing arrays of the BSA-oligonucleotide conjugates on graphitic
carbon surfaces, preferably screen printed carbon ink electrodes.
Surprisingly, we have found that thiol-modified reagents including
thiol-modified oligonucleotides react irreversibly with graphitic
carbon surfaces including screen-printed carbon ink electrodes
(even when not conjugated to a protein such as BSA). Accordingly,
thiol-modified reagents (including thiol modified oligonucleotides
and peptides) can be immobilized on carbon surfaces by incubating
the carbon surfaces with a solution containing the reagents and
allowing the reagent to irreversibly react with the surface.
Furthermore, arrays of such thiol-modified reagents (including
arrays comprising thiol-modified oligonucleotides and/or peptides)
can be formed by printing solutions containing these reagents on
carbon surfaces, incubating the patterned solutions on the surface
and allowing the reagents to irreversibly react with the
surface.
[0183] (v) Assay Devices and Supplementary Reagents
[0184] The methods of the present invention may be used in a
variety of assay devices and/or formats. The assay devices may
include, e.g., assay modules, such as assay plates, cartridges,
multi-well assay plates, reaction vessels, test tubes, cuvettes,
flow cells, assay chips, lateral flow devices, etc., having assay
reagents (which may include targeting agents or other binding
reagents) added as the assay progresses or pre-loaded in the wells,
chambers, or assay regions of the assay module. These devices may
employ a variety of assay formats for specific binding assays,
e.g., immunoassay or immunochromatographic assays. Illustrative
assay devices and formats are described herein below. In certain
embodiments, the methods of the present invention may employ assay
reagents that are stored in a dry state and the assay devices/kits
may further comprise or be supplied with desiccant materials for
maintaining the assay reagents in a dry state. The assay devices
preloaded with the assay reagents can greatly improve the speed and
reduce the complexity of assay measurements while maintaining
excellent stability during storage. The dried assay reagents may be
any assay reagent that can be dried and then reconstituted prior to
use in an assay. These include, but are not limited to, binding
reagents useful in binding assays, enzymes, enzyme substrates,
indicator dyes and other reactive compounds that may be used to
detect an analyte of interest. The assay reagents may also include
substances that are not directly involved in the mechanism of
detection but play an auxiliary role in an assay including, but not
limited to, blocking agents, stabilizing agents, detergents, salts,
pH buffers, preservatives, etc. Reagents may be present in free
form or supported on solid phases including the surfaces of
compartments (e.g., chambers, channels, flow cells, wells, etc.) in
the assay modules or the surfaces of colloids, beads, or other
particulate supports.
[0185] (vi) Measurement Methods
[0186] The methods of the invention can be used with a variety of
methods for measuring the amount of an analyte and, in particular,
measuring the amount of an analyte bound to a solid phase.
Techniques that may be used include, but are not limited to,
techniques known in the art such as cell culture-based assays,
binding assays (including agglutination tests, immunoassays,
nucleic acid hybridization assays, etc.), enzymatic assays,
colorometric assays, etc. Other suitable techniques will be readily
apparent to one of average skill in the art. Some measurement
techniques allow for measurements to be made by visual inspection,
others may require or benefit from the use of an instrument to
conduct the measurement.
[0187] Methods for measuring the amount of an analyte include label
free techniques, which include but are not limited to i) techniques
that measure changes in mass or refractive index at a surface after
binding of an analyte to a surface (e.g., surface acoustic wave
techniques, surface plasmon resonance sensors, ellipsometric
techniques, etc.), ii) mass spectrometric techniques (including
techniques like MALDI, SELDI, etc. that can measure analytes on a
surface), iii) chromatographic or electrophoretic techniques, iv)
fluorescence techniques (which may be based on the inherent
fluorescence of an analyte), etc.
[0188] Methods for measuring the amount of an analyte also include
techniques that measure analytes through the detection of labels
which may be attached directly or indirectly (e.g., through the use
of labeled binding partners of an analyte) to an analyte. Suitable
labels include labels that can be directly visualized (e.g.,
particles that may be seen visually and labels that generate an
measurable signal such as light scattering, optical absorbance,
fluorescence, chemiluminescence, electrochemiluminescence,
radioactivity, magnetic fields, etc). Labels that may be used also
include enzymes or other chemically reactive species that have a
chemical activity that leads to a measurable signal such as light
scattering, absorbance, fluorescence, etc. The use of enzymes as
labels has been well established in Enzyme-Linked ImmunoSorbent
Assays, also called ELISAs, Enzyme ImmunoAssays or EIAs. In the
ELISA format, an unknown amount of antigen is affixed to a surface
and then a specific antibody is washed over the surface so that it
can bind to the antigen. This antibody is linked to an enzyme, and
in the final step a substance is added that the enzyme converts to
a product that provides a change in a detectable signal. The
formation of product may be detectable, e.g., due a difference,
relative to the substrate, in a measurable property such as
absorbance, fluorescence, chemiluminescence, light scattering, etc.
Certain (but not all) measurement methods that may be used with
solid phase binding methods according to the invention may benefit
from or require a wash step to remove unbound components (e.g.,
labels) from the solid phase Accordingly, the methods of the
invention may comprise such a wash step.
[0189] In one embodiment, an analyte(s) of interest in the sample
may be measured using electrochemiluminescence-based assay formats,
e.g. electrochemiluminescence (ECL) based immunoassays. The high
sensitivity, broad dynamic range and selectivity of ECL are
important factors for medical diagnostics. Commercially available
ECL instruments have demonstrated exceptional performance and they
have become widely used for reasons including their excellent
sensitivity, dynamic range, precision, and tolerance of complex
sample matrices. Species that can be induced to emit ECL
(ECL-active species) have been used as ECL labels, e.g., i)
organometallic compounds where the metal is from, for example, the
noble metals of group VIII, including Ru-containing and
Os-containing organometallic compounds such as the
tris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and related
compounds. Species that participate with the ECL label in the ECL
process are referred to herein as ECL coreactants. Commonly used
coreactants include tertiary amines (e.g., see U.S. Pat. No.
5,846,485), oxalate, and persulfate for ECL from RuBpy and hydrogen
peroxide for ECL from luminol (see, e.g., U.S. Pat. No. 5,240,863).
The light generated by ECL labels can be used as a reporter signal
in diagnostic procedures (Bard et al., U.S. Pat. No. 5,238,808,
herein incorporated by reference). For instance, an ECL label can
be covalently coupled to a binding agent such as an antibody,
nucleic acid probe, receptor or ligand; the participation of the
binding reagent in a binding interaction can be monitored by
measuring ECL emitted from the ECL label. Alternatively, the ECL
signal from an ECL-active compound may be indicative of the
chemical environment (see, e.g., U.S. Pat. No. 5,641,623 which
describes ECL assays that monitor the formation or destruction of
ECL coreactants). For more background on ECL, ECL labels, ECL
assays and instrumentation for conducting ECL assays see U.S. Pat.
Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910;
5,641,623; 5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434;
5,786,141; 5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754;
5,240,863; 6,207,369; 6,214,552 and 5,589,136 and Published PCT
Nos. WO99/63347; WO00/03233; WO99/58962; WO99/32662; WO99/14599;
WO98/12539; WO97/36931 and WO98/57154, all of which are
incorporated herein by reference.
[0190] The methods of the invention may be applied to singleplex or
multiplex formats where multiple assay measurements are performed
on a single sample. Multiplex measurements that can be used with
the invention include, but are not limited to, multiplex
measurements i) that involve the use of multiple sensors; ii) that
use discrete assay domains on a surface (e.g., an array) that are
distinguishable based on location on the surface; iii) that involve
the use of reagents coated on particles that are distinguishable
based on a particle property such as size, shape, color, etc.; iv)
that produce assay signals that are distinguishable based on
optical properties (e.g., absorbance or emission spectrum) or v)
that are based on temporal properties of assay signal (e.g., time,
frequency or phase of a signal).
[0191] (vii) Kits
[0192] In one embodiment, the invention provides a kit including a
surface, e.g., multi-well plate or plurality of particles,
comprising a plurality of discrete binding domains each including a
first and second targeting agent, e.g., a first and second
oligonucleotide, and, in a separate vial, container, or
compartment, a first oligonucleotide complement bound to a linking
agent and a second oligonucleotide complement bound to a second
linking agent. In a preferred embodiment, (i) the first targeting
agent and the first targeting agent complement comprise a first
pair of targeting agents, and (ii) the second targeting agent and
the second targeting agent complement comprise a second pair of
targeting agents. Preferably, the first and second pair of
targeting agents comprise a first and second pair of
oligonucleotides, respectively, selected from the pairs of
sequences listed in Table 1.
[0193] The kit further includes, in separate vials, containers, or
compartments, at least 4 oligonucleotides comprising a different
sequence selected from the sequences listed in Table 1(a) and/or
Table 1(b). These sequences may include four sequences selected
from different pairs or may include more than one member of a
pair.
[0194] The kit can include at least 7, 10, 16, or 25 surface bound
oligonucleotides and corresponding oligonucleotide complements. The
oligonucleotides configured for use as targeting agent complements
can be provided pre-bound to a binding reagent such as an antibody
or can be provided modified with a linking agent for attachment to
a binding reagent by the user. Optionally, each oligonucleotide
complement in the kit is coupled to a different binding reagent,
e.g., antibody. The surface-bound oligonucleotides can be
incorporated into an array comprising a plurality of at least 5 (7,
10, 16, or 25) oligonucleotides immobilized to each binding domain
such that a different oligonucleotide sequence is immobilized to a
discrete binding domain. In a specific embodiment, a multi-well
plate can include one or more copies of an oligonucleotide array as
described herein within at least one well of the plate, wherein the
array is positioned on a plurality of binding domains. The plate
can include at least 24, 96, or 384 wells and the array can include
at least 7 oligonucleotides, or at least 10, 16, or 25
oligonucleotides.
[0195] In a specific embodiment, the kit includes a multi-well
plate having one or more copies of an oligonucleotide array within
at least one well(s) of the plate, the array is positioned on a
plurality of binding domains, wherein one or more and optionally,
at least 4 of the binding domains have immobilized thereon a
different oligonucleotide sequence selected from a different
sequence pair from the set of sequence pairs listed in Table 1(a).
In a specific embodiment, the kit includes a multi-well plate
having one or more copies of an oligonucleotide array within at
least one well(s) of the plate, the array is positioned on a
plurality of binding domains, wherein one or more and optionally,
at least 4 of the binding domains have immobilized thereon a
different oligonucleotide sequence selected from a different
sequence pair from the set of sequence pairs listed in Table 1(b).
The kit may further comprise an additional set of oligonucleotides
comprised of two or more oligonucleotides selected from the set of
sequences in Table 1(a), and in a specific embodiment Table 1(b),
wherein the additional oligonucleotides are complementary to the
immobilized oligonucleotides.
[0196] The kit may further include one or more additional
containers, vessels or compartments comprising: (i) a first binding
reagent comprising a first linking agent, wherein the first binding
reagent is specific for a first analyte in the sample, (ii) a first
targeting agent complement comprising a supplemental linking agent,
provided in a separate container, vessel or compartment or as a
component of (i), (iii) a second binding reagent comprising a
second linking agent, wherein the second binding reagent is
specific for a second analyte in the sample, and (iv) a second
targeting agent complement comprising a second supplemental linking
agent, provided in a separate container, vessel or compartment or
as a component of (iii).
[0197] Alternatively, the invention provides a kit for measuring a
plurality of different analytes in a sample, the kit comprising:
(a) a container, vessel or compartment comprising a solid support
including a first targeting agent immobilized to a first region of
the solid support and a second targeting agent immobilized to a
second region of said solid support; and (b) one or more additional
containers, vessels or compartments comprising: (i) a first binding
reagent comprising a first linking agent, wherein said first
binding reagent is specific for a first analyte in said sample,
(ii) a first targeting agent complement comprising a supplemental
linking agent, provided in a separate container, vessel or
compartment or as a component of (b)(i), (iii) a second binding
reagent comprising a second linking agent, wherein said second
binding reagent is specific for a second analyte in said sample,
and (iv) a second targeting agent complement comprising a second
linking agent complement, provided in a separate container, vessel
or compartment or as a component of (b)(iii).
[0198] The invention also contemplates a kit for measuring a
plurality of different analytes in a sample, the kit comprising:
(a) a container, vessel or compartment comprising on a solid
support a first targeting agent immobilized to a first region of
said solid support and a second targeting agent immobilized to a
second region of said solid support; and (b) four or more
additional containers, vessels or compartments comprising: (i) a
first container comprising a first binding reagent comprising a
first linking agent, wherein said first binding reagent is specific
for a first analyte in said sample, (ii) a second container
comprising a first targeting agent complement comprising a
supplemental linking agent, provided in a separate container,
vessel or compartment, (iii) a third container comprising a second
binding reagent comprising a second linking agent, wherein said
second binding reagent is specific for a second analyte in said
sample, and (iv) a fourth container comprising a second targeting
agent complement comprising a second supplemental linking agent,
provided in a separate container, vessel or compartment.
[0199] Still further, the invention provides a kit for measuring a
plurality of different analytes in a sample, the kit comprising:
(a) a container, vessel or compartment comprising on a solid
support a first targeting agent immobilized to a first region of
said solid support and a second targeting agent immobilized to a
second region of said solid support; and (b) two or more additional
containers, vessels or compartments comprising: (i) a first
container comprising a first binding reagent comprising a first
linking agent, wherein said first binding reagent is specific for a
first analyte in said
[0200] sample, and a first targeting agent complement comprising a
supplemental linking agent, and (ii) a second container comprising
a second binding reagent comprising a second linking agent, wherein
said second binding reagent is specific for a second analyte in
said sample, and a second targeting agent complement comprising a
second supplemental linking agent.
[0201] In a specific embodiment, the invention provides a kit
comprising: (a) a multi-well plate comprising a plurality of
discrete binding domains each comprising a first and second
oligonucleotide, respectively, each of said first and second
oligonucleotides are selected from the group consisting of the
sequences listed in Table 1(a) and/or (b).
[0202] The kit can also include instructions for use of the
multi-well plate in a method of conducting a binding assay for a
plurality of analytes, said method comprising the steps of:
[0203] (a) forming a first binding reagent complex comprising a
first binding reagent specific for a first analyte in said
plurality of analytes and said first oligonucleotide, wherein said
first binding reagent is bound to a linking agent and said first
oligonucleotide is bound to a supplemental linking agent wherein
said first binding reagent complex is formed by a reaction between
said linking agent and said supplemental linking agent;
[0204] (b) forming a second binding reagent complex comprising a
second binding reagent specific for a second analyte in said
plurality of analytes and said second oligonucleotide, wherein said
second binding reagent is bound to a second linking agent and said
second oligonucleotide is bound to a second supplemental linking
agent wherein said second binding reagent complex is formed by a
reaction between said second linking agent and said second
supplemental linking agent;
[0205] (c) mixing said first and second binding reagent complexes
with said two or more binding domains each linked to a first
oligonucleotide complement and a second oligonucleotide complement,
respectively, under conditions sufficient to bind said first
oligonucleotide to said first oligonucleotide complement and said
second oligonucleotide to said second oligonucleotide
complement;
[0206] (d) mixing a sample comprising said plurality of analytes to
the mixture formed in step (c);
[0207] (e) adding a plurality of additional binding reagents to the
mixture formed in step (d), wherein said plurality of additional
binding reagents includes (i) a first detection reagent specific
for said first analyte and/or a first binding reagent-first analyte
complex; and (ii) a second detection reagent specific for said
second analyte and/or a second binding reagent-second analyte
complex; and
[0208] (f) measuring the amount of said first and second analytes
bound to said binding domains.
[0209] In one specific embodiment, a multi-well assay plate can be
used to configure an end-user developed assay panel, i.e., an assay
panel built by the end-user with his/her binding reagents to
conduct an assay with the plate. In this embodiment, the end-user
designates which binding reagent is bound to each binding domain. A
multi-well assay plate is provided that includes a plurality of
discrete binding domains including a first binding domain with a
first targeting agent and a second binding domain with a second
targeting agent and, optionally, additional binding domains with
additional targeting agents. Each of the binding domains are
functionalized by the user by selecting individual binding reagents
that will be attached to each of the plurality of binding domains
via a binding reagent complex, as described herein. In a separate
vial, container, or compartment, a set of targeting reagents (each
attached to a linking agent) is provided that includes a first
targeting agent complement, a second targeting agent complement,
and optionally additional targeting agent complements. The first
targeting agent and first targeting agent complement and the second
targeting agent and second targeting agent complement constitute a
first and second pair of targeting agents, respectively. Similarly,
any additional targeting agent complements form pairs with the
different additional targeting agents on the binding domains. In
one preferred embodiment, the targeting agents and targeting agent
complements are oligonucleotides (i.e., an oligonucleotide and its
complement). In this embodiment, the first and second pairs of
targeting agents, and any additional pairs of targeting agents, are
selected from the list of sequences provided in Table 1(a) and/or
Table 1(b).
[0210] Therefore, the user selects which targeting agent/targeting
agent complement will be bound to each specific binding domain. The
user also selects which binding reagent will be bound to each
specific binding domain and forms a binding reagent complex that
includes the targeting agent complement of the targeting agent
attached to the designated binding domain.
[0211] The kit may provide reagents for the users to attach the
supplementary linking agent to the users' binding reagents. When
biotin is the supplementary linking agent, the kit may include
biotin modified with a reactive functional group such as an NHS
ester or hydrazide or maleimide. The plate and/or set of targeting
reagents can further include a labeling kit for attaching a
detectable label to an assay component, such as a detection
reagent. For example, if the multi-well assay plate is configured
to conduct an electrochemiluminescence reaction, the labeling kit
can include a SULFO-TAG.TM. NHS ester, LC-biotin NHS ester, an
optional spin column, and optional labeling buffer solution.
Further provided can be ECL read buffer and optional assay and
antibody diluents.
[0212] The set of targeting reagents preferably includes a quantity
of targeting reagents that matches the number of binding domains
present in the multi-well plate. For example, if the multi-well
plate includes ten discrete binding domains, a set of 10 targeting
reagents are used with that multi-well plate.
[0213] The targeting agents may be provided with a linking agent
that directly binds to the supplementary linking agent, e.g.,
streptavidin or avidin when the supplementary linking agent is
biotin. When the linking agent and supplementary linking agent are
configured to be linked through a bridging agent (e.g., when both
the linking and supplementary linking agents are biotin), the kit
may also provide a bridging reagent solution (e.g., a solution of
streptavidin or avidin) that can be used to attach the binding
reagent to the targeting agent complement. The kit may also provide
a reaction buffer that provides the appropriate conditions for the
linking/bridging reactions and a reaction stop solution. When one
or more of the linking reagents are biotin, the stop solution may
include free biotin to block any unused biotin-binding sites in
streptavidin or avidin that is present as a linking agent,
supplemental linking agent or bridging agent.
[0214] In this embodiment, the user supplies the binding reagents,
e.g., capture and detection antibodies, and designates which
binding reagent will be attached to each of the binding domains.
The binding reagent, e.g., capture antibody, is labeled with a
selected linking agent, e.g., biotin, and attached to a member of a
targeting agent pair via a supplemental linking agent, e.g.,
streptavidin. Meanwhile, the plate is prepared by binding the
targeting agent to the selected binding domain. The modified
binding reagent contacts the surface to form a surface-bound
binding reagent complex that can be used in a subsequent binding
assay for an analyte recognized by the binding reagent. The analyte
of interest is detected by contacting the binding domain with a
labeled binding reagent and measuring the presence of the label
present at that binding domain.
[0215] Alternatively, a multi-well assay plate can be configured
based on a user's specifications, e.g., from a catalog of available
multiplexed assay panels and/or a user can select a set of analytes
to configure a user-customized multiplexed assay for that set of
analytes. A multiplexed assay panel should be selected and
optimized such that individual assays function well together. For
example, the sample may require dilution prior to being assayed.
Sample dilutions for specific sample matrices of interest are
optimized for a given panel to minimize sample matrix effects and
to maximize the likelihood that all the analytes in the panel will
be within the dynamic range of the assay. In a preferred
embodiment, all of the analytes in the panel are analyzed with the
same sample dilution in at least one sample type. In another
preferred embodiment, all of the analytes in a panel are measured
using the same dilution for most sample types.
[0216] For a given immunoassay panel, the detection antibody
concentration and the number of detectable labels per protein (L/P
ratio) for the detection antibody are adjusted to bring the
expected levels of all analytes into a quantifiable range at the
same sample dilution. If one wants to increase the high end of the
quantifiable range for a given analyte, then the L/P can be
decreased and/or the detection antibody concentration is decreased.
On the other hand, if one wants to increase the lower end of the
quantifiable range, the L/P can be increased, the detection
antibody concentration can be increased if it is not at the
saturation level, and/or the background signal can be lowered.
[0217] Calibration standards for use with an assay panel are
selected to provide the appropriate quantifiable range with the
recommended sample dilution for the panel.
[0218] The calibration standards have known concentrations of one
of more of the analytes in the panel. Concentrations of the
analytes in unknown samples are determined by comparison to these
standards. In one embodiment, calibration standards comprise
mixtures of the different analytes measured by an assay panel.
Preferably, the analyte levels in a combined calibrator are
selected such that the assay signals for each analyte are
comparable, e.g., within a factor of two, a factor of five or a
factor of 10. In another embodiment, calibration standards include
mixtures of analytes from multiple different assay panels.
[0219] A calibration curve may be fit to the assay signals measured
with calibration standards using, e.g., curve fits known in the art
such as linear fits, 4-parameter logistic (4-PL) and 5-parameter
(5-PL) fits. Using such fits, the concentration of analytes in an
unknown sample may be determined by backfitting the measured assay
signals to the calculated fits. Measurements with calibration
standards may also be used to determine assay characteristics such
as the limit of detection (LOD), limit of quantification (LOQ),
dynamic range, and limit of linearity (LOL).
[0220] As part of a multiplexed panel development, assays are
optimized to reduce calibrator and detection antibody non-specific
binding. In sandwich immunoassays, specificity mainly comes from
capture antibody binding. Some considerations for evaluating
multiplexed panels include: (a) detection antibody non-specific
binding to capture antibodies is reduced to lower background of
assays in the panel, and this can be achieved by adjusting the
concentrations and L/P of the detection antibodies; (b)
non-specific binding of detection antibodies to other calibrators
in the panel is also undesirable and should be minimized; (c)
non-specific binding of other calibrators in the panel and other
related analytes should be minimized; if there is calibrator
non-specific binding, it can reduce the overall specificity of the
assays in the panel and it can also yield unreliable results as
there will be calibrator competition to bind the capture
antibody.
[0221] In one specific embodiment, the kit further includes one or
more reagents to insure that the correct steps of the assay
protocol were followed. In addition, the variability of the assay
steps can be measured independent of the assay. In this embodiment,
a series of complementary oligonucleotides diluted in diluents used
to run an assay are provided in the kit. As the correct steps are
performed, the oligonucleotides in the various diluents bind,
extending the chain. The final diluent can include the final
complementary oligonucleotide with a reporter or label to provide a
detectable indicator of the successful processing of the assay. For
example, a first binding domain of a multi-well plate is coated
with a BSA-oligonucleotide having sequence A. the assay diluent
includes an oligonucleotide sequence complementary to sequence A,
A', and an additional sequence, B. A and B do not interact with one
another and specifically interact only with their complements. The
final diluent contains a supplemental oligonucleotide comprising
the complement of oligonucleotide B, B', and a detectable label.
Therefore, the detectable signal from the first binding domain can
be used to verify that the sample was added or the detection moiety
was added. The signal generated can also be used to detect if the
correct volumes and concentrations of reagents were added, as well
as whether there was variability in sample handling procedures
and/or equipment. The oligonucleotide chain used in the process can
include multiple overlapping sequences.
[0222] Different assays in the panel may require different
incubation times and sample handling requirements for optimal
performance. Therefore, the goal is to select a protocol that's
optimized for most assays in the panel. Optimization of the assay
protocol includes, but is not limited to, adjusting one or more of
the following protocol parameters: timing (incubation time of each
step), preparation procedure (calibrators, samples, controls,
etc.), and number of wash steps.
[0223] In a further specific embodiment, the methods described in
FIG. 5(c) can be carried out using a kit configured for use in an
instrument as described in U.S. application Ser. No. 12/844,440,
the disclosure of which is incorporated herein by reference. The
apparatus described therein enables fully automated random access
analysis of samples using array-based multiplexed multi-well plate
consumables. The apparatus achieves enhanced sensitivity and high
sample throughput. All the biological reagents required for an
assay are added by the user and/or provided in the apparatus prior
to processing, thereby minimizing the consumable and reagent
requirements for the apparatus. Thus, the method illustrated in
FIG. 5(c) can be carried out in an apparatus as disclosed in U.S.
Ser. No. 12/844,440 by providing the reagents required for the
method in a kit that includes a test plate, e.g., as illustrated in
FIG. 4(a)-(b) of U.S. Ser. No. 12/844,440, or as described herein
as a multi-well assay plate, that includes the surface bearing
targeting agent-modified binding domains, as well as (i) a set of
one or more binding reagents modified by supplemental linking
agents, (ii) a set of one or more targeting agent complements
modified by linking agents; (iii) a set of detection reagents for
the analytes of interest; and optionally (iv) a set of control
reagents and/or (v) a set of calibrators. The test plate can be
mixed with kit components (i) and (ii), mixed with sample, and then
detection reagents are added. Alternatively, components (i) and
(ii) can be provided pre-mixed and directly combined with the
surface, or components (i) and (ii) can be provided in the kit as
separate components that are mixed in solution by the instrument
and then combined with the surface as described above. Any surface
binding step can optionally be followed by a washing step to remove
any unbound components of the assay before proceeding to the next
step. In a specific example, components (i) and (ii) are provided
pre-mixed and added to the test plate in the instrument. The test
plate is incubated, washed, the sample is optionally pre-diluted,
and then added to the test plate. The test plate is incubated
again, washed, and detection reagents are added. The test plate is
incubated once more, washed, read buffer is added, and signal is
detected by the instrument indicating the relative presence and/or
absence of analyte(s) in the sample. The reagents can be provided
in any suitable container in the kit, including test tubes or
Eppendorf tubes, or in dedicated sections of an auxiliary plate as
described in U.S. Ser. No. 12/844,440.
[0224] These and other embodiments of the invention are illustrated
in the following non-limited examples.
EXAMPLES
Example 1
Direct Assay Format
[0225] The procedure for the preparation and use of a multi-well
plate for a direct assay is illustrated in FIG. 2. The experimental
layout, i.e., which oligonucleotide sequence was located in which
binding domain of a multi-well assay plate, was noted. The
multi-well assay plate was obtained from Meso Scale Discovery, a
division of Meso Scale Diagnostics, LLC (Rockville, Md.). A working
solution of each individual oligonucleotide sequence complement
(550 uL) was prepared by diluting a stock solution of sequence
complement about 50 times in Diluent 100 (stock solutions of
oligonucleotide sequence complement and Diluent 100 are available
from Meso Scale Discovery).
[0226] Each capture antibody was labeled with an oligonucleotide
having a terminal thiol group using a bifunctional coupling reagent
(sulfosuccinimidyl 4-(N-maleimidomethyl)-1-cyclohexane carboxylate
("SMCC")) and conventional coupling protocols as shown in FIG.
5(d), e.g., protein is reacted with the NHS-ester in SMCC to label
the protein and the resulting complex is reacted with thiolated
oligonucleotides which reacts with the maleimide group in SMCC. A
pooled solution (50 uL) of a set of antibody-oligo-conjugates was
then added to each well of the multi-well plate in hybridization
buffer for 1 hour at room temperature to hybridize the
complementary oligonucleotide sequences and thereby immobilize the
capture antibodies to the multi-well plate to form a plurality of
binding reagent complexes.
[0227] A solution including a plurality of analytes (25 uL of MSD
Diluent 2, with 25 uL calibrator solution of MSD Diluent 2) was
added to each well of the prepared plate, incubated for 1 hour at
room temperature, washed 3.times.PBS, and a set of labeled
detection antibodies (50 uL of MSD Diluent 3) was added to each
well of the multi-well plate. The plate was incubated with shaking
and the wells were washed with 3.times.PBS, filled with 150 uL of
Read Buffer T (Meso Scale Discovery) and analyzed on a SECTOR.RTM.
Imager instrument.
[0228] This protocol was used to conduct an assay for a 7-plex
chemokine panel and the result are shown in FIG. 8(a)-(g). The
analytes assayed in this experiment were Eotaxin, MIP-1b, TARC,
IL-8, MCP-4, IP-10, and MCP-1 (all human analytes). In addition,
this protocol was used to conduct an assay for a 10-plex TH1/TH2
panel including IFNg, IL-1b, IL-2, IL-4, IL-5, IL-8, IL-10,
IL-12p70, IL-13, and TNFa (all human analytes). The results for
both assays were compared with a standard direct immunoassay absent
oligonucleotides linkers.
Example 2
Indirect Assay Format Using an Oligonucleotide-SA Conjugate
[0229] The procedure for the preparation and use of a multi-well
plate for an indirect assay using oligonucleotide-SA conjugates is
illustrated in FIG. 9(a)-(c). The experimental layout for the
multi-well assay plate was noted as in Example 1. A working
solution of each individual oligonucleotide sequence complement
bound to streptavidin (SA) (550 uL) was prepared by diluting a
stock solution of sequence complement about 50 times in Diluent 100
(stock solutions of oligonucleotide sequence complement and Diluent
100 are available from Meso Scale Discovery). A solution of
biotinylated antibody was added to the desired working solution of
oligonucleotide sequence complement to prepare a set of individual
biotinylated capture antibody/oligonucleotide-SA mixtures. The
concentration of biotinylated antibody in the mixture was in the
range of about 5-30 ug/mL The mixture was gently mixed for 30-45
minutes at room temperature. Fifty (50) uL of biotin solution (Meso
Scale Discovery) (approximately a three-fold excess of biotin) was
added to each individual biotinylated antibody/oligonucleotide
complement mixture and this mixture was gently mixed for 10-15
minutes at room temperature. Equal volumes (550 uL) of individual
biotinylated antibody/oligonucleotide complements were combined and
the total volume of the solution was adjusted to 5500 uL with the
addition of a Conjugation Buffer (PBS with 0.1M EDTA at pH
7.4).
[0230] The multi-well plate was allowed to warm to room temperature
(approximately 10 minutes). Fifty (50) uL of biotinylated
antibodies/oligonucleotide complements was added to each well of
the plate. The plate was covered with an adhesive seal and
incubated for 1 hour on a plate shaker at room temperature. Each
well was washed with phosphate buffered saline (PBS, 3.times.).
[0231] A solution including a plurality of analytes (25 uL of MSD
Diluent 2, with 25 uL calibrator solution of MSD Diluent 2) was
added to each well of the prepared plate, incubated for 1 hour at
room temperature, washed 3.times.PBS, and a set of labeled
detection antibodies (50 uL of MSD Diluent 3) was added to each
well of the multi-well plate. The plate was incubated with shaking
and the wells were washed with 3.times.PBS, filled with 150 uL of
Read Buffer T (Meso Scale Discovery) and analyzed on a SECTOR.RTM.
Imager instrument.
Example 3
Indirect Assay Format Using Neat SA/Biotinylated
Oligonucleotides
[0232] The procedure for the preparation and use of a multi-well
plate for an indirect assay using biotinylated capture antibodies,
neat streptavidin, and biotinylated oligonucleotides is illustrated
in FIG. 10(a)-(c). The experimental layout for the multi-well assay
plate was noted as in Example 1. A working solution of an excess of
each individual oligonucleotide sequence complement bound to
streptavidin (SA) (550 uL) was prepared by diluting a stock
solution of sequence complement about 50 times in Diluent 100
(stock solutions of oligonucleotide sequence complement and Diluent
100 are available from Meso Scale Discovery). A solution of
biotinylated antibody was added to the desired working solution of
oligonucleotide sequence complement to prepare a set of individual
biotinylated capture antibody/oligonucleotide-SA mixtures. The
concentration of biotinylated antibody in the mixture was in the
range of about 5-30 ug/mL The mixture was gently mixed for 30-45
minutes at room temperature. Fifty (50) uL of biotin solution (Meso
Scale Discovery) (approximately a three-fold excess of biotin) was
added to each individual biotinylated antibody/oligonucleotide
complement mixture and this mixture was gently mixed for 10-15
minutes at room temperature. Equal volumes (550 uL) of individual
biotinylated antibody/oligonucleotide complements were combined and
the total volume of the solution was adjusted to 5500 uL with the
addition of a Conjugation Buffer (NEED COMPOSITION; available from
Meso Scale Discovery).
[0233] The multi-well plate was allowed to warm to room temperature
(approximately 10 minutes). Fifty (50) uL of biotinylated
antibodies/oligonucleotide complements was added to each well of
the plate. The plate was covered with an adhesive seal and
incubated for 1 hour on a plate shaker at room temperature. Each
well was washed with phosphate buffered saline (PBS, 3.times.).
[0234] A solution including a plurality of analytes (25 uL of MSD
Diluent 2, with 25 uL calibrator solution of MSD Diluent 2) was
added to each well of the prepared plate, incubated for 1 hour at
room temperature, washed 3.times.PBS, and a set of labeled
detection antibodies (50 uL of MSD Diluent 3) was added to each
well of the multi-well plate. The plate was incubated with shaking
and the wells were washed with 3.times.PBS, filled with 150 uL of
Read Buffer T (Meso Scale Discovery) and analyzed on a SECTOR.RTM.
Imager instrument.
Example 4
Comparative Results of Three Assay Formats Using 7-Plex Cytokine B
Panel
[0235] A 7-plex cytokine B panel (IL-8, hTNF-a, hEotaxin-3,
h-Eotaxin, hMCP-1, HIP-10 and hMIP-1a) was tested in the assay
formats described in Examples 1, 2, and 3, i.e. direct, indirect
with oligonucleotide-SA conjugate and indirect with neat
SA/biotinylated oligonucleotides. LOD values for assays were
estimated from an 8-point calibration curve assume 2.5 standard
deviations. The results are shown in FIG. 11(a)-(g). The LOD values
for direct and indirect assay formats compare with LOD values
observed for standard passive adsorption and immunoassay formats
for all tested assays. The indirect approach with neat SA showed
significant spread in LOD values: some assays, IL-8 and hIP-10,
showed higher LOD values compared to the remaining assay formats,
while Eotaxin and MIP-10 showed lower LOD values.
[0236] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the method in addition to those described herein
will become apparent to those skilled in the art from the foregoing
description and accompanying figures. Such modifications are
intended to fall within the scope of the claims. Various
publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.
Sequence CWU 1
1
50112DNAArtificial Sequenceoligonucleotide 1acatcggtag tt
12212DNAArtificial Sequenceoligonucleotide 2aactaccgat gt
12312DNAArtificial Sequenceoligonucleotide 3acgtcccagt tg
12412DNAArtificial Sequenceoligonucleotide 4caactgggac gt
12512DNAArtificial Sequenceoligonucleotide 5agaagaagat cc
12612DNAArtificial Sequenceoligonucleotide 6ggatcttctt ct
12712DNAArtificial Sequenceoligonucleotide 7aggttcagtg ca
12812DNAArtificial Sequenceoligonucleotide 8tgcactgaac ct
12912DNAArtificial Sequenceoligonucleotide 9atcaggatac gc
121012DNAArtificial Sequenceoligonucleotide 10gcgtatcctg at
121112DNAArtificial Sequenceoligonucleotide 11atcattacca cc
121212DNAArtificial Sequenceoligonucleotide 12ggtggtaatg at
121312DNAArtificial Sequenceoligonucleotide 13attaacggga gc
121412DNAArtificial Sequenceoligonucleotide 14gctcccgtta at
121512DNAArtificial Sequenceoligonucleotide 15cagaggtctt aa
121612DNAArtificial Sequenceoligonucleotide 16ttaagacctc tg
121712DNAArtificial Sequenceoligonucleotide 17caggtgtcca tt
121812DNAArtificial Sequenceoligonucleotide 18aatggacacc tg
121912DNAArtificial Sequenceoligonucleotide 19catccaatcc ag
122012DNAArtificial Sequenceoligonucleotide 20ctggattgga tg
122112DNAArtificial Sequenceoligonucleotide 21cctacgatat ac
122212DNAArtificial Sequenceoligonucleotide 22gtatatcgta gg
122312DNAArtificial Sequenceoligonucleotide 23cgaatgtaga gt
122412DNAArtificial Sequenceoligonucleotide 24actctacatt cg
122512DNAArtificial Sequenceoligonucleotide 25cggtttgaga ta
122612DNAArtificial Sequenceoligonucleotide 26tatctcaaac cg
122712DNAArtificial Sequenceoligonucleotide 27cttacaacgc ca
122812DNAArtificial Sequenceoligonucleotide 28tggcgttgta ag
122912DNAArtificial Sequenceoligonucleotide 29ctttctcggc ac
123012DNAArtificial Sequenceoligonucleotide 30gtgccgagaa ag
123112DNAArtificial Sequenceoligonucleotide 31gacataaagc ga
123212DNAArtificial Sequenceoligonucleotide 32tcgctttatg tc
123312DNAArtificial Sequenceoligonucleotide 33gccatagtct ct
123412DNAArtificial Sequenceoligonucleotide 34agagactatg gc
123512DNAArtificial Sequenceoligonucleotide 35gctaattcac ca
123612DNAArtificial Sequenceoligonucleotide 36tggtgaatta gc
123712DNAArtificial Sequenceoligonucleotide 37ggtcgtgttt ca
123812DNAArtificial Sequenceoligonucleotide 38tgaaacacga cc
123912DNAArtificial Sequenceoligonucleotide 39gttgattctg tc
124012DNAArtificial Sequenceoligonucleotide 40gacagaatca ac
124112DNAArtificial Sequenceoligonucleotide 41tacccggaat aa
124212DNAArtificial Sequenceoligonucleotide 42ttattccggg ta
124312DNAArtificial Sequenceoligonucleotide 43tgcttgactt gg
124412DNAArtificial Sequenceoligonucleotide 44ccaagtcaag ca
124512DNAArtificial Sequenceoligonucleotide 45ttccacttag gg
124612DNAArtificial Sequenceoligonucleotide 46ccctaagtgg aa
124712DNAArtificial Sequenceoligonucleotide 47ttgtctagcg gc
124812DNAArtificial Sequenceoligonucleotide 48gccgctagac aa
124912DNAArtificial Sequenceoligonucleotide 49tttcccttgc ta
125012DNAArtificial Sequenceoligonucleotide 50tagcaaggga aa 12
* * * * *