Nucleic Acid Binding Immuno-Sandwich Assay (NULISA)

Abstract

Disclosed herein are highly sensitive immunoassays that utilize a capture / release mechanism to reduce nonspecific binding and achieve detection at attomolar levels of sensitivity. Also disclosed herein are kits that can be used to perform these highly sensitive immunoassays.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 943,135, filed December 3, 2019, the disclosure of which is incorporated herein by reference in its entirety. (Sequence Listing) This specification is submitted with a computer readable form (CRF) copy of the Sequence Listing. The CRF, entitled 14582-003-228_SEQ_LISTING.txt, created on December 1, 2020, is 13,423 bytes in size and is incorporated herein by reference in its entirety. (1. Field) The present invention relates to the field of molecular biology. Specifically, the present disclosure relates to highly sensitive immunoassays for the detection of target biomolecules or molecular complexes. [Background technology]

[0002] (2.Background) The detection of minute amounts of target molecules or molecular complexes in biological samples is of great importance for both scientific research and clinical studies. Thanks to inventions and advances in polymerase chain reaction (PCR), tremendous progress has been made in the detection and analysis of nucleic acids over the past few decades. However, the poor performance of current immunoassays for the detection of other biomolecules, such as proteins, remains a technological bottleneck for many important applications. For example, early cancer detection is estimated to require effective detection of analytes at 4,000 molecules / mL or ∼7 attomolar concentrations ("aM"), which exceeds the capabilities of existing immunoassay technologies. Therefore, there is a great need to improve the performance of immunoassays. The present disclosure provides highly sensitive immunoassays that address these needs and offer related advantages. Summary of the Invention

[0003] (3. Summary of the Invention) Embodiment 1. An assay method for detecting an analyte in a sample, comprising: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (3) generating a reporter from the immune complex based on the proximity between the first target label and the second target label; and (4) detecting the reporter, thereby detecting the analyte. : An assay method comprising:

[0004]

[0022] Embodiment 2. The assay method of embodiment 1, wherein (i) the first target label comprises a first identification barcode ("ID") ("target ID") that is analyte-specific; (ii) the second target label comprises a second target ID; or (iii) both (i) and (ii).

[0005]

[0022] Embodiment 3. The assay method of embodiment 1 or 2, wherein (i) the reporter comprises a first target identifier; (ii) the reporter comprises a second target identifier; or (iii) both (i) and (ii).

[0006] Embodiment 4. An assay method for detecting an analyte in a sample, comprising: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (3) generating a reporter from the immune complex, wherein the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and (4) detecting the reporter, thereby detecting the analyte. : An assay method comprising:

[0007]

[0033] Embodiment 5. The assay method of embodiment 4, wherein the reporter is generated from the immune complex based on proximity between the first target label and the second target label.

[0008]

[0032] Embodiment 6. The assay method of any one of embodiments 1 to 5, wherein the reporter is a nucleic acid reporter.

[0009] Embodiment 7. (i) the first target ID in the reporter is the complementary sequence of the first target ID in the first binding moiety; (ii) the second target ID in the reporter is the complementary sequence of the second target ID in the first binding moiety; (iii) Both (i) and (ii) :An assay method according to embodiment 6.

[0010] Embodiment 8. The assay method of any one of embodiments 1 to 7, further comprising a step (2a) between steps (2) and (3): releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first acceptor group, wherein step (2a) is before step (3), after step (3), or simultaneous with step (3).

[0011]

[0033] Embodiment 9. The assay method of embodiment 8, wherein the second binding moiety further comprises a second presentation group.

[0012] Embodiment 10. Step 2(b) between step 2(a) and step (3): (2b) introducing a second solid surface and recapturing the immune complex to the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; The assay method of embodiment 9, further comprising:

[0013] Embodiment 11. Step 2(c) between step 2(b) and step (3) (2c) washing the second solid surface to remove unbound molecules; The assay method of embodiment 10, further comprising:

[0014] Embodiment 12. The assay method of embodiment 10 or 11, further comprising step (2d): releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second acceptor group.

[0015] Embodiment 13. The assay method of embodiment 12, wherein step (2d) is before step (3), after step (3), or simultaneous with step (3).

[0016]

[0033] Embodiment 14. The assay method of any one of embodiments 2 to 13, wherein (i) the first target ID and the second target ID are identical; or (ii) the first target ID and the second target ID are different.

[0017]

[0033] Embodiment 15. The assay method of any one of the preceding embodiments, further comprising step (2e): binding a sample label comprising an ID that is sample-specific ("sample ID") to (i) the first target label, (ii) the second target label, or (iii) both the first target label and the second target label.

[0018]

[0033] Embodiment 16. The assay method of embodiment 15, wherein the reporter formed in each sample comprises a sample ID.

[0019]

[0033] Embodiment 17. The assay method of any one of the preceding embodiments, wherein the first presentation group is a polypeptide fused to the first binder, a polynucleotide conjugated to said first binder, or a chemical compound conjugated to said first binder.

[0020]

[0033] Embodiment 18. The assay method of any one of embodiments 9 to 17, wherein the second presentation group is a polypeptide fused to a second binder, a polypeptide conjugated to a second binder, or a chemical compound conjugated to said second binder.

[0021]

[0033] Embodiment 19. The assay method of any one of the preceding embodiments, further comprising releasing the reporter from the immune complex.

[0022]

[0033] Embodiment 20. The assay method of any one of embodiments 6 to 19, wherein step (4) further comprises PCR amplification of the nucleic acid reporter.

[0023]

[0033] Embodiment 21. The assay method of any one of embodiments 6 to 20, further comprising purifying the nucleic acid reporter.

[0024]

[0033] Embodiment 22. The assay method of any one of the preceding embodiments, wherein step (1) comprises forming the immune complexes in solution prior to capturing the immune complexes on the first solid surface.

[0025]

[0033] Embodiment 23. The assay method of any one of embodiments 1 to 21, wherein step (1) comprises pre-capturing the first binder on the first solid surface before an immune complex is formed on the first solid surface.

[0026] Embodiment 24. The assay method of any one of embodiments 1 to 21, wherein in step (1), the immune complexes are formed in solution and simultaneously captured on the first solid surface.

[0027] Embodiment 25. (i) whether the first binder binds directly to the analyte and the second binder binds directly to the analyte; (ii) the first binder binds directly to the analyte and the second binder binds indirectly to the analyte; (iii) the first binder indirectly binds the analyte and the second binder directly binds the analyte; or (iv) the first binder indirectly binds the analyte and the second binder indirectly binds the analyte; 10. The assay method of any one of the preceding embodiments.

[0028] Embodiment 26. (i) whether the first binder binds to a first primary antibody or fragment thereof that directly binds to the analyte; (ii) the second binder binds to a second primary antibody or fragment thereof that directly binds to the analyte; or (iii) Both (i) and (ii): 10. The assay method of any one of the preceding embodiments.

[0029] Embodiment 27. (i) whether the first and second binders bind to non-interfering epitopes on the analyte; (ii) the first and second binders bind to non-overlapping epitopes on the analyte; or (iii) the first and second binders bind to different epitopes on the analyte: The assay method of any one of embodiments 1 to 25.

[0030] Embodiment 28. (i) at least one additional recapture cycle between steps (2) and (3), comprising: releasing the immune complex from the solid surface on which it is captured, recapturing the immune complex onto an additional solid surface coupled to a first acceptor group, and washing the additional solid surface to remove unbound molecules; (ii) at least one additional recapture cycle between steps (2c) and (2d), comprising: releasing the immune complex from the solid surface on which it is captured, recapturing the immune complex onto a further solid surface coupled to the first acceptor group or the second acceptor group, and washing the further solid surface to remove unbound molecules; or (iii) Both (i) and (ii) 10. The assay method of any one of the preceding embodiments, further comprising:

[0031]

[0042] Embodiment 29. The assay method of any one of embodiments 8 to 28, wherein any of the releasing is by increasing the temperature to 70°C.

[0032]

[0033] Embodiment 30. The assay method of any one of embodiments 1-29, wherein (i) the first presenting group is attached to the first acceptor group via a thioester group, a disulfide bond, or a cleavable bond; (ii) the second presenting group is attached to the second acceptor group via a thioester group, a disulfide bond, or a cleavable bond; or both (i) and (ii).

[0033] Embodiment 31. The assay method of embodiment 30, wherein (i) the first presenting group is attached to the first acceptor group via a photocleavable, chemically cleavable, or enzymatically cleavable bond; (ii) the second presenting group is attached to the second acceptor group via a photocleavable, chemically cleavable, or enzymatically cleavable bond; or both (i) and (ii).

[0034] Embodiment 32. The assay method of any one of embodiments 1-29, wherein (i) the first presenting group binds to the first acceptor group via a protein-protein interaction; (ii) the second presenting group binds to the second acceptor group via a protein-protein interaction; or both (i) and (ii).

[0035] Embodiment 33. The assay method of any one of embodiments 1-29, wherein (i) the first presenting group binds to the first acceptor group via streptavidin or biotin on avidin; (ii) the second presenting group binds to the second acceptor group via biotin on streptavidin or avidin; or both (i) and (ii).

[0036] Embodiment 34. (i) the first presenting group is a first nucleic acid tag ("first tag") and the first accepting group is a first nucleic acid capture probe ("first probe"); or (ii) the second presenting group is a second nucleic acid tag ("second tag") and the second accepting group is a second nucleic acid capture probe ("second probe"); The assay method of any one of embodiments 1 to 29.

[0037] Embodiment 35. (i) the first presenting group is a first nucleic acid tag ("first tag") and the first accepting group is a first nucleic acid capture probe ("first probe"); and (ii) the second presenting group is a second nucleic acid tag ("second tag") and the second accepting group is a second nucleic acid capture probe ("second probe"). The assay method of any one of embodiments 9 to 29.

[0038] Embodiment 36. (i) whether the first probe is a protein that specifically binds to the first tag; (ii) whether the first probe is a protein and nucleic acid complex that specifically binds to the first tag; (iii) the first probe is a nucleic acid molecule, wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; or (iv) the first probe is a nucleic acid molecule, wherein the first probe or a fragment thereof hybridizes to the first tag or a fragment thereof: The assay method of embodiment 34 or 35.

[0039] Embodiment 37. (i) whether the second probe is a protein that specifically binds to the second tag; (ii) the second probe is a protein and nucleic acid complex that specifically binds to the second tag; (iii) the second probe is a nucleic acid molecule, wherein the second probe or a fragment thereof is complementary to the second tag or a fragment thereof; or (iv) the second probe is a nucleic acid molecule, wherein the second probe or a fragment thereof hybridizes to the second tag or a fragment thereof: An assay method according to any one of embodiments 34 to 36.

[0040] Embodiment 38. The assay method of embodiment 36 or 37, wherein the complementarity is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementarity.

[0041]

[0042] Embodiment 39. The assay method of any one of embodiments 34 to 38, wherein (i) the first probe is directly coupled to the first solid surface; or (ii) the second probe is directly coupled to the second solid surface.

[0042]

[0046] Embodiment 40. The assay method of any one of embodiments 34-38, wherein (i) the first probe is directly coupled to the first solid surface; and (ii) the second probe is directly coupled to the second solid surface.

[0043]

[0042] Embodiment 41. The assay method of any one of embodiments 34 to 38, wherein (i) the first probe hybridizes to a universal probe that is directly coupled to a first solid surface; or (ii) the second probe hybridizes to a universal probe that is directly coupled to a second solid surface.

[0044]

[0042] Embodiment 42. The assay method of any one of embodiments 34-38, wherein (i) the first probe hybridizes to a universal probe that is directly coupled to a first solid surface; and (ii) the second probe hybridizes to a universal probe that is directly coupled to a second solid surface.

[0045]

[0042] Embodiment 43. The assay method of any one of embodiments 34-38, wherein (i) the first probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to the first solid surface; or (ii) the second probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to the second solid surface.

[0046]

[0042] Embodiment 44. The assay method of any one of embodiments 34-38, wherein (i) the first probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to a first solid surface; and (ii) the second probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to a second solid surface.

[0047]

[0044] Embodiment 45. The assay method of any one of embodiments 34 to 44, wherein in step (1), the first tag and the second tag are cooperatively captured to a first solid surface.

[0048]

[0046] Embodiment 46. The assay method of embodiment 45, wherein a first fragment of a first probe is complementary to a first tag or a fragment thereof, and a second fragment of said first probe is complementary to a second tag or a fragment thereof, wherein said complementary regions comprise the unconjugated ends of said first and said second tags.

[0049]

[0046] Embodiment 47. The assay method of embodiment 45, wherein the contiguous fragment of the first probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first tag and second tag are ligated to form a linked nucleic acid, the junction region of the linked nucleic acid is complementary to the contiguous fragment of the first probe.

[0050]

[0044] Embodiment 48. The assay method of embodiment 45, wherein a first fragment of a first probe is complementary to a first tag or a fragment thereof, and another second fragment of said first probe is complementary to a second tag or a fragment thereof; and said complementary regions do not include the unconjugated ends of said first and said second tags.

[0051]

[0046] Embodiment 49. The assay method of embodiment 45, wherein the first solid surface is coupled to both the first probe and an additional nucleic acid probe, and the additional probe, or fragment thereof, is complementary to the first tag or fragment thereof.

[0052]

[0046] Embodiment 50. The assay method of any one of embodiments 35 to 49, wherein the first tag and the second tag are cooperatively captured to a second solid surface in step (2b).

[0053]

[0044] Embodiment 51. The assay method of embodiment 50, wherein the first fragment of the second probe is complementary to the first tag or a fragment thereof, and the second fragment of the second probe is complementary to the second tag or a fragment thereof, wherein the complementary regions comprise the unconjugated ends of the first and second tags.

[0054] Embodiment 52. The assay method of embodiment 50, wherein the contiguous fragment of the second probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first tag and second tag are ligated to form a linked nucleic acid, the junction region of the linked nucleic acid is complementary to the contiguous fragment of the second probe.

[0055]

[0044] Embodiment 53. The assay method of embodiment 50, wherein a first fragment of the second probe is complementary to a first tag or a fragment thereof, and another second fragment of said second probe is complementary to a second tag or a fragment thereof; and said complementary regions do not include the unconjugated ends of said first and said second tags.

[0056] Embodiment 54. The assay method of embodiment 50, wherein the second solid surface is coupled to both the second probe and an additional nucleic acid probe, and the additional probe, or fragment thereof, is complementary to the second tag or fragment thereof.

[0057]

[0033] Embodiment 55. The assay method of any one of embodiments 34 to 54, wherein (i) the complementary fragment of the first tag and the first probe consists of 10 to 30 base pairs; (ii) the complementary fragment of the second tag and the second probe consists of 10 to 30 base pairs; or both (i) and (ii).

[0058]

[0033] Embodiment 56. The assay method of any one of embodiments 34 to 55, wherein (i) the complementary fragment of the first tag and the first probe consists of 20 to 30 base pairs; (ii) the complementary fragment of the second tag and the second probe consists of 20 to 30 base pairs; or both (i) and (ii).

[0059]

[0046] Embodiment 57. The assay method of any one of embodiments 34 to 56, wherein (i) the first tag comprises an A- and / or T-rich sequence and the first probe comprises a complementary A- and / or T-rich sequence; (ii) the second tag comprises an A- and / or T-rich sequence and the second probe comprises a complementary A- and / or T-rich sequence; or both (i) and (ii).

[0060]

[0044] Embodiment 58. The assay method of embodiment 57, wherein the A- and / or T-rich sequence has a short length such that the binding from the complementary A- and / or T-rich sequence is weaker than both the binding between the first binder and the analyte and the binding between the second binder and the analyte, thereby maintaining the immune complex stable in releasing steps (2a) and / or (2d).

[0061]

[0046] Embodiment 59. The assay method of any one of the preceding embodiments, wherein the analyte is a binding pair of two molecules; wherein a first binder binds to one molecule of the binding pair and a second binder binds to the other molecule of the binding pair.

[0062]

[0044] Embodiment 60. The assay method of any one of the preceding embodiments, wherein the analyte is a nucleic acid and the first and second binders of the nucleic acid analyte comprise nucleic acids that are complementary to different fragments of the nucleic acid analyte.

[0063]

[0044] Embodiment 61. The assay method of any one of embodiments 1 or 59, wherein the analyte is a peptide or protein, and (i) the first binder is an antibody or antibody fragment that specifically binds to the analyte; (ii) the second binder is an antibody or antibody fragment that specifically binds to the analyte; or both (i) and (ii).

[0064]

[0044] Embodiment 62. The assay method of any one of the preceding embodiments, wherein the sample is a serum sample or a plasma sample.

[0065] Embodiment 63. (i) in step (3), the immune complex is captured on the first solid surface while the nucleic acid reporter is generated; or (ii) In step (3), after the immune complex is released from the first solid surface, the nucleic acid reporter is generated: An assay method according to any one of embodiments 6 to 62.

[0066] Embodiment 64. (i) in step (3), the immune complex is captured on a second solid surface while the nucleic acid reporter is generated; or (ii) In step (3), after the immune complex is released from the second solid surface, the nucleic acid reporter is generated: An assay method according to any one of embodiments 10 to 62.

[0067] Embodiment 65. The assay method of any one of embodiments 1 to 64, wherein (i) the first target label is directly bound to the first binder; or (ii) said first target label is indirectly bound to said first binder.

[0068] Embodiment 66. The assay method of any one of embodiments 1 to 65, wherein (i) the second target label is directly bound to the second binder; or (ii) said second target label is indirectly bound to said second binder.

[0069] Embodiment 67. (i) whether the first target label is conjugated to the first binder; (ii) the first target label is non-covalently bound to the first binder; (iii) the first target label is conjugated to a first presentation group; (iv) the first target label is non-covalently bound to the first presentation group; or (v) the first target label is part of the first presentation group: The assay method of any one of embodiments 1 to 66.

[0070] Embodiment 68. (i) whether a second target label is conjugated to a second binder; (ii) the second target label is non-covalently bound to the second binder; (iii) the second target label is conjugated to a second presentation group; (iv) the second target label is non-covalently bound to the second presentation group; or (v) the second target label is part of the second presentation group: An assay method according to any one of embodiments 1 to 67.

[0071] Embodiment 69. (i) the first presenting group is directly attached to the first binder; or (ii) the first presenting group is indirectly attached to the first binder: The assay method of any one of embodiments 1 to 68.

[0072] Embodiment 70. (i) the second presenting group is directly attached to the second binder; or (ii) the second presenting group is indirectly attached to the second binder: The assay method of any one of embodiments 9 to 69.

[0073] Embodiment 71. (i) whether the first presenting group is conjugated to the first binder; (ii) the first presenting group is non-covalently bound to the first binder; (iii) the first presentation group is conjugated to a first target label; (iv) the first presentation group is non-covalently bound to the first target label; or (v) the first presentation group is part of the first target label: An assay method according to any one of embodiments 1 to 70.

[0074] Embodiment 72. (i) whether the second presentation group is conjugated to the second binder; (ii) the second presenting group is non-covalently bound to the second binder; (iii) the second presentation group is conjugated to a second target label; (iv) the second presentation group is non-covalently bound to the second target label; or (v) the second presentation group is part of the second target label: The assay method of any one of embodiments 9 to 71.

[0075] Embodiment 73. (i) whether the first target label is a nucleic acid molecule; (ii) the second target label is a nucleic acid molecule; or (iii) Both (i) and (ii): The assay method of any one of embodiments 1 to 72.

[0076] Embodiment 74. (i) whether the first target label hybridizes with the first tag; (ii) a second target label hybridizes to a second tag; or (iii) Both (i) and (ii): An assay method according to any one of embodiments 34 to 73.

[0077]

[0044] Embodiment 75. The assay method of any one of embodiments 15 to 74, wherein the sample label is a single-stranded nucleic acid molecule ("single-stranded sample label").

[0078]

[0044] Embodiment 76. The assay method of any one of embodiments 15 to 74, wherein the sample label is a double-stranded nucleic acid molecule ("double-stranded sample label").

[0079] Embodiment 77. The sample label (i) a double-stranded nucleic acid molecule containing two 5' overhangs; (ii) a double-stranded nucleic acid molecule containing two 3' overhangs; (iii) a double-stranded nucleic acid molecule comprising a 5' overhang and a 3' overhang; (iv) a double-stranded nucleic acid molecule comprising a 5' overhang and a blunt end; or (v) a double-stranded nucleic acid molecule containing a 3' overhang and a blunt end An assay method according to embodiment 76, wherein:

[0080] Embodiment 78. The sample label (i) hybridizing to a first target label via the overhang of the sample label; (ii) hybridizing, via the overhang of the sample label, to a second target label; or (iii) Both (i) and (ii): The assay method of embodiment 77.

[0081] Embodiment 79. Step (3) comprises: (a) generating the nucleic acid reporter by linking a first tag and a second tag, and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second tag; (b) generating the nucleic acid reporter by linking the first tag and a surrogate nucleic acid of the second tag (the "second surrogate"), and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second surrogate; (c) generating the nucleic acid reporter by ligating a surrogate nucleic acid of the first tag (a "first surrogate") to the second tag, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second tag; or (d) linking the first surrogate and the second surrogate to generate the nucleic acid reporter, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second surrogate; Contains: wherein the first tag or fragment thereof is complementary to the first surrogate or fragment thereof, and the second tag or fragment thereof is complementary to the second surrogate or fragment thereof; An assay method according to any one of embodiments 34 to 78.

[0082] Embodiment 80. The method of claim 80, wherein step (3) comprises: (a) generating a nucleic acid reporter by linking a first tag and a second target label, and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second target label; (b) generating the nucleic acid reporter by ligating the first tag and a surrogate nucleic acid of the second target label (the "second surrogate"), and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second surrogate; (c) generating the nucleic acid reporter by ligating a surrogate nucleic acid of the first tag ("first surrogate") to the second target label, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second target label; or (d) linking the first surrogate and the second surrogate to generate the nucleic acid reporter, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second surrogate; Contains: wherein said first tag or fragment thereof is complementary to said first surrogate or fragment thereof, and said second target label or fragment thereof is complementary to said second surrogate or fragment thereof; An assay method according to any one of embodiments 34 to 78.

[0083] Embodiment 81. The method of claim 81, wherein step (3) comprises: (a) linking a first target label and a second tag to generate a nucleic acid reporter, and detecting the nucleic acid reporter composed of a fragment of the first target label and a fragment of the second tag; (b) generating the nucleic acid reporter by ligating the first target label and a surrogate nucleic acid of the second tag (the "second surrogate"), and detecting the nucleic acid reporter composed of a fragment of the first target label and a fragment of the second surrogate; (c) generating the nucleic acid reporter by ligating a surrogate nucleic acid of the first target label (a "first surrogate") to the second tag, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second tag; or (d) linking the first surrogate and the second surrogate to generate the nucleic acid reporter, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second surrogate; Contains: wherein said first target label or fragment thereof is complementary to said first surrogate or fragment thereof, and said second tag or fragment thereof is complementary to said second surrogate or fragment thereof; An assay method according to any one of embodiments 34 to 78.

[0084] Embodiment 82. Step (3) comprises: (a) generating a nucleic acid reporter by linking a first target label and a second target label, and detecting the nucleic acid reporter composed of a fragment of the first target label and a fragment of the second target label; (b) generating the nucleic acid reporter by ligating the first target label and a surrogate nucleic acid of the second target label (the "second surrogate"), and detecting the nucleic acid reporter composed of a fragment of the first target label and a fragment of the second surrogate; (c) generating the nucleic acid reporter by ligating a surrogate nucleic acid of the first target label (a "first surrogate") to the second target label, and detecting the nucleic acid reporter comprised of a fragment of the first surrogate and a fragment of the second target label; or (d) linking the first surrogate and the second surrogate to generate the nucleic acid reporter, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second surrogate; Contains: wherein the first target label or fragment thereof is complementary to the first surrogate or fragment thereof, and the second target label or fragment thereof is complementary to the second surrogate or fragment thereof; An assay method according to any one of embodiments 34 to 78.

[0085] Embodiment 83. The linking comprises: (i) (a) a first tag or a first surrogate thereof, and (b) a second tag or a second surrogate thereof, and (c) a surrogate nucleic acid of a single-stranded sample label (a "sample surrogate") or one strand of a double-stranded sample label; (ii) (a) the first tag or a first surrogate thereof, (b) a second target label or a second surrogate thereof, and (c) a surrogate nucleic acid of the single-stranded sample label (a "sample surrogate") or one strand of the double-stranded sample label; (iii) (a) a first target label or a first surrogate thereof, and (b) the second tag or a second surrogate thereof, and (c) a surrogate nucleic acid of the single-stranded sample label (a "sample surrogate") or one strand of the double-stranded sample label; or (iv) (a) the first target label or a first surrogate thereof, (b) the second target label or a second surrogate thereof, and (c) a surrogate nucleic acid of the single-stranded sample label (a "sample surrogate") or one strand of the double-stranded sample label. : including concatenating; wherein the sample label or fragment thereof is complementary to the sample surrogate or fragment thereof; An assay method according to any one of embodiments 79 to 82.

[0086]

[0033] Embodiment 84. The assay method of embodiment 83, wherein in each of (i) to (iv), linking comprises linking (c) between (a) and (b).

[0087]

[00444] Embodiment 85. The assay method of any one of embodiments 79-84, wherein the nucleic acid reporter is formed by proximity ligation.

[0088]

[00444] Embodiment 86. The assay method of any one of embodiments 79-84, wherein the nucleic acid reporter is formed by proximity extension.

[0089]

[0042] Embodiment 87. The assay method of any one of embodiments 79-86, wherein the nucleic acid reporter comprises (a) a first target ID or a surrogate nucleic acid for the first target ID (a "first target ID surrogate"), (b) a second target ID or a surrogate nucleic acid for the second target ID (a "second target ID surrogate"), and (c) a sample ID.

[0090]

[0042] Embodiment 88. The assay method of any one of embodiments 2-87, comprising simultaneously detecting at least two analytes in a sample by simultaneously detecting a unique target ID associated with each analyte.

[0091] Embodiment 89. The assay method of embodiment 88, comprising in step (1) proportionally reducing the signal from at least one of the analytes by adding a non-functional binder to the solution, wherein the non-functional binder competes with the first binder for binding to the analyte but is either unconjugated or conjugated to a presenting group that does not bind to the first acceptor group.

[0092]

[0046] Embodiment 90. The assay method of any one of embodiments 2 to 89, wherein in step (4), detecting the analyte comprises simultaneous detection of the first target ID and the second target ID.

[0093] Embodiment 91. The assay method of any one of the preceding embodiments, further comprising in step (1) mixing a reference analyte.

[0094]

[0044] Embodiment 92. The assay method of embodiment 91, wherein the reference analyte is an analyte that is not present in the sample.

[0095]

[0044] Embodiment 93. The assay method of embodiment 91 or 92, wherein the reference analyte is a protein, nucleic acid, or chemical compound not present in the sample.

[0096]

[0044] Embodiment 94. The assay method of embodiment 93, wherein the reference analyte is a viral protein, a bacterial protein, or an insect protein.

[0097]

[0046] Embodiment 95. The assay method of any one of embodiments 15-94, comprising simultaneously detecting the analyte in at least two samples by simultaneously detecting a unique sample ID in a nucleic acid reporter associated with each sample.

[0098]

[0044] Embodiment 96. The assay method of embodiment 95, further comprising pooling the nucleic acid reporters from at least two samples prior to or concurrently with the detecting in step (4).

[0099]

[0042] Embodiment 97. The assay method of any one of embodiments 15-95, comprising simultaneously detecting at least two analytes in at least two samples by simultaneously detecting a unique sample ID and a unique target ID in a nucleic acid reporter associated with each analyte in each sample.

[0100]

[0044] Embodiment 98. The assay method of embodiment 97, further comprising pooling the nucleic acid reporters for at least two analytes from at least two samples prior to or concurrently with the detecting in step (4).

[0101]

[00444] Embodiment 99. The assay method of any one of the preceding embodiments, wherein the nucleic acid reporters are detected by multiplexed qPCR, multiplexed digital PCR, or NGS.

[0102]

[0044] Embodiment 100. The assay method of any one of the preceding embodiments, wherein the nucleic acid reporter is detected by NGS.

[0103]

[0042] Embodiment 101. The assay method of any one of embodiments 91-100, wherein the detecting further comprises normalizing the reporter generated from the sample analyte to a reporter generated from a reference analyte.

[0104] Embodiment 102. The assay method of any one of the preceding embodiments, wherein (i) the first solid surface is selected from the group consisting of a magnetic particle surface and a well of a microtiter plate; (ii) the second solid surface is selected from the group consisting of a magnetic particle surface and a well of a microtiter plate; or both (i) and (ii).

[0105]

[0062] Embodiment 103. An assay method for detecting an analyte in a sample, comprising: (1) mixing a first binding moiety comprising a first binder and a first presentation group, a second binding moiety comprising a second binder and a second presentation group, and a sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface by disrupting the bond between the first presenting group and the first accepting group; (4) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; (6) binding a sample label comprising a sample-specific ID (“sample ID”) to (i) the first target label, (ii) the second target label, or (iii) both the first target label and the second target label; (7) generating a nucleic acid reporter from the immune complex based on proximity between the first target label and the second target label, wherein the nucleic acid reporter comprises the first target ID, the second target ID, and the sample ID; (8) releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group; and (9) detecting the nucleic acid reporter by qPCR, thereby detecting the analyte. : An assay method comprising:

[0106] Embodiment 104. (1) mixing a first binding moiety comprising a first binder and a first presentation group, a second binding moiety comprising a second binder and a second presentation group, and a sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface by disrupting the bond between the first presenting group and the first accepting group; (4) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; (6) binding a sample label comprising a sample-specific ID (“sample ID”) to (i) the first target label, (ii) the second target label, or (iii) both the first target label and the second target label; (7) generating a nucleic acid reporter from the immune complex based on proximity between the first target label and the second target label, wherein the nucleic acid reporter comprises the first target ID, the second target ID, and the sample ID; (8) pooling nucleic acid reporters from at least two samples; (9) releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group; (9) amplifying the nucleic acid reporter; (10) purifying the nucleic acid reporter; and (11) Detecting the nucleic acid reporter by next-generation sequencing (NGS), thereby detecting the analyte. 1. An assay method for detecting an analyte in at least two samples, comprising:

[0107] Embodiment 105. (i) a first binding moiety comprising a first binder, a first presentation group, and a first target label; (ii) a second binding moiety comprising a second binder and a second target label; and (iii) the first acceptor Includes; And here, (i) the first and second binders bind to epitopes on the analyte; and (ii) the first presenting group binds to the first accepting group; A system for detecting an analyte in a sample.

[0108]

[0066] Embodiment 106. The system of embodiment 105, wherein (i) the first target label comprises a first identification barcode ("ID") ("target ID") that is analyte-specific; (ii) the second target label comprises a second target ID; or (iii) both (i) and (ii).

[0109] Embodiment 107. (i) a first binding moiety comprising a first binder, a first presentation group, and a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific; (ii) a second binding moiety comprising a second binder and a second target label comprising a second target ID; and (iii) the first acceptor and wherein (i) the first and second binders bind to epitopes on the analyte; and (ii) the first presenting group binds to the first accepting group; A system for detecting an analyte in a sample.

[0110] Embodiment 108. The system of any one of embodiments 105 to 107, wherein the reporter is a nucleic acid reporter.

[0111]

[0062] Embodiment 109. The system of any one of embodiments 105-108, wherein the second binding moiety further comprises a second presenting group, wherein the system further comprises a second acceptor group, and the second presenting group binds to said second acceptor group.

[0112]

[0044] Embodiment 110. The system of any one of embodiments 106-109, wherein (i) the first target ID and the second target ID are identical; or (ii) the first target ID and the second target ID are different.

[0113]

[0062] Embodiment 111. The system of any one of embodiments 105-110, further comprising a sample label comprising an ID ("sample ID") that is sample-specific, wherein the sample label binds to (i) the first target label, (ii) the second target label, or (iii) both the first target label and the second target label.

[0114]

[00444] Embodiment 112. The system of any one of embodiments 106-111, further comprising reagents for proximity ligation or proximity extension to generate a nucleic acid reporter comprising (i) a first target ID and a second target ID, or (ii) said first target ID, said second target ID, and a sample ID.

[0115] Embodiment 113. The system of any one of embodiments 105 to 112, further comprising a first solid surface.

[0116] Embodiment 114. The system of any one of embodiments 109 to 113, further comprising a second solid surface.

[0117] Embodiment 115. The system of embodiment 113 or 114, wherein (i) the first solid surface is a magnetic particle surface or a well of a microtiter plate; (ii) the second solid surface is a magnetic particle surface or a well of a microtiter plate; or both (i) and (ii).

[0118] Embodiment 116. The system of embodiment 114 or 115, wherein a first solid surface is coupled to a first acceptor group and a second solid surface is coupled to a second acceptor group.

[0119]

[0062] Embodiment 117. The system of any one of embodiments 105 to 116, wherein the first presentation group is a polypeptide fused to the first binder, a polynucleotide conjugated to said first binder, or a chemical compound conjugated to said first binder.

[0120]

[0062] Embodiment 118. The system of any one of embodiments 109 to 117, wherein the second presentation group is a polypeptide fused to a second binder, a polypeptide conjugated to a second binder, or a chemical compound conjugated to said second binder.

[0121]

[0062] Embodiment 119. The system of any one of embodiments 108 to 118, further comprising reagents for PCR amplification of the nucleic acid reporter.

[0122]

[0062] Embodiment 120. The system of any one of embodiments 108 to 119, further comprising a reagent for purifying the nucleic acid reporter.

[0123] Embodiment 121. (i) whether the first binder binds directly to the analyte and the second binder binds directly to the analyte; (ii) the first binder binds directly to the analyte and the second binder binds indirectly to the analyte; (iii) the first binder indirectly binds the analyte and the second binder directly binds the analyte; or (iv) the first binder indirectly binds the analyte and the second binder indirectly binds the analyte; A system described in any one of embodiments 105 to 120.

[0124] Embodiment 122. (i) whether the first binder binds to a first primary antibody or fragment thereof that directly binds to the analyte; (ii) the second binder binds to a second primary antibody or fragment thereof that directly binds to the analyte; or (iii) Both (i) and (ii): A system described in any one of embodiments 105 to 121.

[0125] Embodiment 123. (i) whether the first and second binders bind to non-interfering epitopes on the analyte; (ii) the first and second binders bind to non-overlapping epitopes on the analyte; or (iii) the first and second binders bind to different epitopes on the analyte: A system described in any one of embodiments 105 to 122.

[0126]

[0062] Embodiment 124. The system of any one of embodiments 105-123, wherein (i) the first presenting group is linked to the first acceptor group via a thioester group, a disulfide bond, or a cleavable bond; (ii) the second presenting group is linked to the second acceptor group via a thioester group, a disulfide bond, or a cleavable bond; or both (i) and (ii).

[0127] Embodiment 125. The system of embodiment 124, wherein (i) the first presenting group is attached to the first acceptor group via a photocleavable, chemically cleavable, or enzymatically cleavable bond; (ii) the second presenting group is attached to the second acceptor group via a photocleavable, chemically cleavable, or enzymatically cleavable bond; or both (i) and (ii).

[0128] Embodiment 126. The system of any one of embodiments 105 to 123, wherein (i) the first presenting group binds to the first acceptor group via a protein-protein interaction; (ii) the second presenting group binds to the second acceptor group via a protein-protein interaction; or both (i) and (ii).

[0129] Embodiment 127. The system of any one of embodiments 105 to 123, wherein (i) the first presenting group is bound to the first acceptor group via streptavidin or biotin on avidin; (ii) the second presenting group is bound to the second acceptor group via biotin on streptavidin or avidin; or both (i) and (ii).

[0130] Embodiment 128. (i) the first presenting group is a first nucleic acid tag ("first tag") and the first accepting group is a first nucleic acid capture probe ("first probe"); or (ii) the second presenting group is a second nucleic acid tag ("second tag") and the second accepting group is a second nucleic acid capture probe ("second probe"); A system described in any one of embodiments 105 to 123.

[0131] Embodiment 129. The system of any one of embodiments 109 to 123, wherein (i) the first presentation group is a first nucleic acid tag ("first tag") and the first acceptor group is a first nucleic acid capture probe ("first probe"); and (ii) the second presentation group is a second nucleic acid tag ("second tag") and the second acceptor group is a second nucleic acid capture probe ("second probe").

[0132] Embodiment 130. (i) whether the first probe is a protein that specifically binds to the first tag; (ii) whether the first probe is a protein and nucleic acid complex that specifically binds to the first tag; (iii) the first probe is a nucleic acid molecule, wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; or (iv) the first probe is a nucleic acid molecule, wherein the first probe or a fragment thereof hybridizes to the first tag or a fragment thereof; 130. The system of embodiment 128 or 129.

[0133] Embodiment 131. (i) whether the second probe is a protein that specifically binds to the second tag; (ii) the second probe is a protein and nucleic acid complex that specifically binds to the second tag; (iii) the second probe is a nucleic acid molecule, wherein the second probe or a fragment thereof is complementary to the second tag or a fragment thereof; or (iv) the second probe is a nucleic acid molecule, wherein the second probe or a fragment thereof hybridizes to the second tag or a fragment thereof: 131. The system of embodiment 128 or 130.

[0134] Embodiment 132. The system of embodiment 130 or 131, wherein the complementarity is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementarity.

[0135] Embodiment 133. The system of any one of embodiments 128 to 132, wherein (i) the first probe is directly coupled to the first solid surface; or (ii) the second probe is directly coupled to the second solid surface.

[0136] Embodiment 134. The system of any one of embodiments 128 to 132, wherein (i) the first probe is directly coupled to the first solid surface; and (ii) the second probe is directly coupled to the second solid surface.

[0137] Embodiment 135. The system of any one of embodiments 128 to 132, wherein (i) the first probe hybridizes to a universal probe that is directly coupled to a first solid surface; or (ii) the second probe hybridizes to a universal probe that is directly coupled to a second solid surface.

[0138] Embodiment 136. The system of any one of embodiments 128 to 132, wherein (i) the first probe hybridizes to a universal probe that is directly coupled to a first solid surface; and (ii) the second probe hybridizes to a universal probe that is directly coupled to a second solid surface.

[0139] Embodiment 137. The system of any one of embodiments 128 to 132, wherein (i) the first probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to a first solid surface; or (ii) the second probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to a second solid surface.

[0140] Embodiment 138. The system of any one of embodiments 128 to 132, wherein (i) the first probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to a first solid surface; and (ii) the second probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to a second solid surface.

[0141] Embodiment 139. The system of any one of embodiments 128 to 138, wherein the first tag and the second tag are cooperatively captured to the first solid surface.

[0142]

[0066] Embodiment 140. The system of embodiment 139, wherein a first fragment of a first probe is complementary to a first tag or a fragment thereof, and a second fragment of said first probe is complementary to a second tag or a fragment thereof, wherein said complementary regions comprise the unconjugated ends of said first and said second tags.

[0143]

[0062] Embodiment 141. The system of embodiment 139, wherein the contiguous fragment of the first probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first tag and second tag are ligated to form a linked nucleic acid, the junction region of the linked nucleic acid is complementary to the contiguous fragment of the first probe.

[0144]

[0062] Embodiment 142. The system of embodiment 139, wherein a first fragment of a first probe is complementary to a first tag or a fragment thereof, and another second fragment of said first probe is complementary to a second tag or a fragment thereof; and said complementary regions do not include the unconjugated ends of said first and said second tags.

[0145] Embodiment 143. The system of embodiment 139, wherein the first solid surface is coupled to both the first probe and an additional nucleic acid probe, and wherein said additional probe, or fragment thereof, is complementary to the first tag or fragment thereof.

[0146] Embodiment 144. The system of any one of embodiments 128 to 143, wherein the first tag and the second tag are cooperatively captured to a second solid surface.

[0147]

[0066] Embodiment 145. The system of embodiment 144, wherein the first fragment of the second probe is complementary to the first tag or a fragment thereof, and the second fragment of the second probe is complementary to the second tag or a fragment thereof, wherein said complementary regions comprise the unconjugated ends of the first and second tags.

[0148]

[0062] Embodiment 146. The system of embodiment 144, wherein the continuous fragment of the second probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first tag and second tag are ligated to form a linked nucleic acid, the junction region of the linked nucleic acid is complementary to the continuous fragment of the second probe.

[0149] Embodiment 147. The system of embodiment 144, wherein a first fragment of the second probe is complementary to a first tag or a fragment thereof, and another second fragment of said second probe is complementary to a second tag or a fragment thereof; and said complementary regions do not include the unconjugated ends of said first and said second tags.

[0150] Embodiment 148. The system of embodiment 144, wherein the second solid surface is coupled to both the second probe and an additional nucleic acid probe, and said additional probe, or fragment thereof, is complementary to the second tag or fragment thereof.

[0151]

[0062] Embodiment 149. The system of any one of embodiments 128 to 148, wherein (i) the complementary fragment of the first tag and the first probe consists of 10 to 30 base pairs; (ii) the complementary fragment of the second tag and the second probe consists of 10 to 30 base pairs; or both (i) and (ii).

[0152] Embodiment 150. The system of any one of embodiments 128 to 149, wherein (i) the complementary fragment of the first tag and the first probe consists of 20 to 30 base pairs; (ii) the complementary fragment of the second tag and the second probe consists of 20 to 30 base pairs; or both (i) and (ii).

[0153]

[0066] Embodiment 151. The system of any one of embodiments 128 to 150, wherein (i) the first tag comprises an A- and / or T-rich sequence and the first probe comprises a complementary A- and / or T-rich sequence; (ii) the second tag comprises an A- and / or T-rich sequence and the second probe comprises a complementary A- and / or T-rich sequence; or both (i) and (ii).

[0154]

[0062] Embodiment 152. The system of embodiment 151, wherein the A and / or T rich sequence has a short length such that the binding from the complementary A and / or T rich sequence is weaker than both the binding between the first binder and the analyte and the binding between the second binder and the analyte.

[0155]

[0062] Embodiment 153. The system of any one of embodiments 105-152, wherein the analyte is a binding pair of two molecules; wherein a first binder binds to one molecule of the binding pair and a second binder binds to the other molecule of the binding pair.

[0156]

[0062] Embodiment 154. The system of any one of embodiments 105 to 153, wherein the analyte is a nucleic acid and the first and second binders of the nucleic acid analyte comprise nucleic acids that are complementary to different fragments of the nucleic acid analyte.

[0157]

[0062] Embodiment 155. The system of any one of embodiments 105-153, wherein the analyte is a peptide or protein, and (i) the first binder is an antibody or antibody fragment that specifically binds to the analyte; (ii) the second binder is an antibody or antibody fragment that specifically binds to the analyte; or both (i) and (ii).

[0158] Embodiment 156. The system of any one of embodiments 105 to 155, wherein the sample is a serum sample or a plasma sample.

[0159] Embodiment 157. The system of any one of embodiments 105 to 156, wherein (i) the first target label is directly bound to the first binder; or (ii) the first target label is indirectly bound to the first binder.

[0160] Embodiment 158. The system of any one of embodiments 105 to 157, wherein (i) the second target label is directly bound to the second binder; or (ii) said second target label is indirectly bound to said second binder.

[0161] Embodiment 159. (i) whether the first target label is conjugated to the first binder; (ii) the first target label is non-covalently bound to the first binder; (iii) the first target label is conjugated to a first presentation group; (iv) the first target label is non-covalently bound to the first presentation group; or (v) the first target label is part of the first presentation group: A system described in any one of embodiments 105 to 158.

[0162] Embodiment 160. (i) whether a second target label is conjugated to a second binder; (ii) the second target label is non-covalently bound to the second binder; (iii) the second target label is conjugated to a second presentation group; (iv) the second target label is non-covalently bound to the second presentation group; or (v) the second target label is part of the second presentation group: A system described in any one of embodiments 105 to 159.

[0163] Embodiment 161. The system of any one of embodiments 105-160, wherein (i) the first presenting group is directly attached to the first binder; or (ii) the first presenting group is indirectly attached to the first binder.

[0164] Embodiment 162. The system of any one of embodiments 109 to 161, wherein (i) the second presenting group is directly attached to the second binder; or (ii) the second presenting group is indirectly attached to the second binder.

[0165] Embodiment 163. (i) whether the first presenting group is conjugated to the first binder; (ii) the first presenting group is non-covalently bound to the first binder; (iii) the first presentation group is conjugated to a first target label; (iv) the first presentation group is non-covalently bound to the first target label; or (v) the first presentation group is part of the first target label: A system described in any one of embodiments 105 to 162.

[0166] Embodiment 164. (i) whether the second presentation group is conjugated to the second binder; (ii) the second presenting group is non-covalently bound to the second binder; (iii) the second presentation group is conjugated to a second target label; (iv) the second presentation group is non-covalently bound to the second target label; or (v) the second presentation group is part of the second target label: A system described in any one of embodiments 109 to 163.

[0167] Embodiment 165. (i) whether the first target label is a nucleic acid molecule; (ii) the second target label is a nucleic acid molecule; or (iii) Both (i) and (ii): A system described in any one of embodiments 105 to 164.

[0168] Embodiment 166. (i) whether the first target label hybridizes with the first tag; (ii) a second target label hybridizes to a second tag; or (iii) Both (i) and (ii): A system described in any one of embodiments 128 to 165.

[0169]

[0066] Embodiment 167. The system of any one of embodiments 106 to 166, wherein the sample label is a single-stranded nucleic acid molecule ("single-stranded sample label").

[0170]

[0066] Embodiment 168. The system of any one of embodiments 106 to 166, wherein the sample label is a double-stranded nucleic acid molecule ("double-stranded sample label").

[0171] Embodiment 169. The sample label (i) a double-stranded nucleic acid molecule containing two 5' overhangs; (ii) a double-stranded nucleic acid molecule containing two 3' overhangs; (iii) a double-stranded nucleic acid molecule comprising a 5' overhang and a 3' overhang; (iv) a double-stranded nucleic acid molecule comprising a 5' overhang and a blunt end; or (v) a double-stranded nucleic acid molecule comprising a 3' overhang and a blunt end: The system of embodiment 168, wherein

[0172] Embodiment 170. The sample label (i) hybridizing to a first target label via the overhang of the sample label; (ii) hybridizing, via the overhang of the sample label, to a second target label; or (iii) Both (i) and (ii): The system described in embodiment 169.

[0173]

[0066] Embodiment 171. The system of any one of embodiments 106-170, wherein the system is capable of simultaneously detecting at least two analytes in a sample by simultaneously detecting a unique target ID associated with each analyte.

[0174] Embodiment 172. The system of any one of embodiments 105 to 171, wherein the system further comprises a reference analyte.

[0175] Embodiment 173. The system of embodiment 172, wherein the reference analyte is an analyte that is not present in the sample.

[0176]

[0062] Embodiment 174. The system of embodiment 172 or 173, wherein the reference analyte is a protein, nucleic acid, or chemical compound not present in the sample.

[0177]

[0062] Embodiment 175. The system of embodiment 174, wherein the reference analyte is a viral protein, a bacterial protein, or an insect protein.

[0178]

[0062] Embodiment 176. The system of any one of embodiments 111-175, wherein the system is capable of simultaneously detecting an analyte in at least two samples by simultaneously detecting a unique sample ID in a nucleic acid reporter associated with each sample.

[0179]

[0062] Embodiment 177. The system of any one of embodiments 110-176, wherein the system is capable of simultaneously detecting at least two analytes in at least two samples by simultaneously detecting a unique sample ID and a unique target ID in a nucleic acid reporter associated with each analyte in each sample.

[0180]

[0062] Embodiment 178. The system of any one of embodiments 105 to 177, comprising reagents and / or equipment for multiplexed qPCR, multiplexed digital PCR, or NGS for detecting nucleic acid reporters.

[0181]

[0062] Embodiment 179. The system of any one of embodiments 105 to 177, comprising reagents and / or equipment for NGS to detect the nucleic acid reporter.

[0182] Embodiment 180. The system of any one of embodiments 105 to 179, wherein the system is comprised in a kit.

[0183] Embodiment 181. The system of embodiment 180, wherein the kit comprises a binding buffer, an immobilization buffer, a wash buffer, a release buffer, or any combination thereof.

[0184] Embodiment 182. A method for detecting a target protein comprising: (1) a first binding moiety comprising a first binder, a first presentation group, and a first target label comprising a first identification barcode ("ID") ("target ID") that is analyte-specific; (2) a second binding moiety comprising a second binder, a second presentation group, and a second target label comprising a second target ID; (3) a first acceptor group, a first solid surface, a second acceptor group, and a second solid surface; (4) reagents for ligation and a sample label comprising an ID ("sample ID") that is sample-specific; and (5) reagents for quantitative PCR; wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the first accepting group is coupled to the first solid surface and configured to capture the first presenting group; (iii) the second accepting group is coupled to the second solid surface and configured to capture the second presenting group; (iv) the first target label is directly or indirectly bound to the first binder, and the second target label is directly or indirectly bound to the second binder; (v) the first presenting group is directly or indirectly attached to the first binder, and the second presenting group is directly or indirectly attached to the second binder; and (vi) the sample label binds to both the first target label and the second target label; A system for detecting an analyte in a sample.

[0185] Embodiment 183. The system of embodiment 182, wherein the system is comprised in a kit.

[0186] Embodiment 184. The system of embodiment 183, wherein the kit further comprises a binding buffer, an immobilization buffer, a wash buffer, a release buffer, or any combination thereof.

[0187] Embodiment 185. A ligation system comprising: (1) a first binding moiety comprising a first binder, a first presentation group, and a first target label comprising a first identification barcode ("ID") ("target ID") that is analyte-specific; (2) a second binding moiety comprising a second binder, a second presentation group, and a second target label comprising a second target ID; (3) a first acceptor group, a first solid surface, a second acceptor group, and a second solid surface; and (4) a reagent for ligation and a sample label comprising an ID ("sample ID") that is sample-specific; wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the first accepting group is coupled to the first solid surface and configured to capture the first presenting group; (iii) the second accepting group is coupled to the second solid surface and configured to capture the second presenting group; (iv) the first target label is directly or indirectly bound to the first binder, and the second target label is directly or indirectly bound to the second binder; (v) the first presenting group is directly or indirectly bound to the first binder, and the second presenting group is directly or indirectly bound to the second binder; and (vi) the sample label binds to both the first target label and the second target label; A system for detecting an analyte in a sample.

[0188] Embodiment 186. The system of embodiment 185, wherein the system is comprised in a kit.

[0189] Embodiment 187. The system of embodiment 186, wherein the kit comprises a binding buffer, an immobilization buffer, a wash buffer, a release buffer, or any combination thereof.

[0190] Provided herein are assay methods that use two capture binders. In some embodiments, provided herein are assay methods for detecting an analyte in a sample, comprising: (1) mixing a first binder, a second binder, and the sample in solution, wherein the first and second binders bind to non-interfering epitopes on the analyte and form immune complexes, which are captured on the first solid surface in contact with the solution via binding between a first presenting group conjugated to the first binder and a first accepting group coupled to a first surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface by disrupting the bond between the first presenting group and the first accepting group; (4) introducing a second solid surface and recapturing the immune complex via binding between a second presenting group conjugated to the second binder and a second accepting group coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. An assay method comprising:

[0191] In some embodiments, step (1) comprises forming an immune complex in solution before capturing the immune complex on a first solid surface. In some embodiments, step (1) comprises pre-capturing a first binder on a first solid surface before capturing the immune complex on the first solid surface. In some embodiments, in step (1), an immune complex is formed in solution and simultaneously captured on a first solid surface.

[0192] In some embodiments, the assay methods provided herein further comprise at least one additional recapture cycle between steps (5) and (6), comprising: releasing the immune complex from the solid surface on which it is captured, recapturing the immune complex onto an additional solid surface coupled to the first acceptor group or the second acceptor group, and washing the additional solid surface to remove unbound molecules.

[0193] In some embodiments, (i) the first presenting group is linked to the first accepting group via a thioester group, a disulfide bond, or a cleavable bond; (ii) the second presenting group is linked to the second accepting group via a thioester group, a disulfide bond, or a cleavable bond; or both (i) and (ii).

[0194] In some embodiments, (i) the first presenting group is attached to the first acceptor group via a photocleavable, chemically cleavable, or enzymatically cleavable bond; (ii) the second presenting group is attached to the second acceptor group via a photocleavable, chemically cleavable, or enzymatically cleavable bond; or both (i) and (ii).

[0195] In some embodiments, (i) the first presenting group is a first nucleic acid tag ("first tag") and the first accepting group is a first nucleic acid capture probe ("first probe"), wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; or (ii) the second presenting group is a second nucleic acid tag ("second tag") and the second accepting group is a second nucleic acid capture probe ("second probe"), wherein the second probe or a fragment thereof is complementary to the second tag or a fragment thereof.

[0196] In some embodiments, (i) the first presenting group is a first tag and the first accepting group is a first probe; and (ii) the second presenting group is a second tag and the second accepting group is a second probe.

[0197] In some embodiments, (i) the first probe is directly coupled to a first solid surface; (ii) the second probe is directly coupled to a second solid surface; or both (i) and (ii).

[0198] In some embodiments, (i) the first probe hybridizes to a universal probe that is directly coupled to a first solid surface; (ii) the second probe hybridizes to a universal probe that is directly coupled to a second solid surface; or both (i) and (ii).

[0199] In some embodiments, (i) the first probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to a first solid surface; (ii) the second probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to a second solid surface; or both (i) and (ii).

[0200] In some embodiments, the first tag and the second tag are cooperatively captured to a first solid surface in step (1).

[0201] In some embodiments, a first fragment of a first probe is complementary to a first tag or a fragment thereof, and a second fragment of the first probe is complementary to a second tag or a fragment thereof, wherein the complementary regions comprise the unconjugated ends of the first and second tags.

[0202] In some embodiments, the contiguous fragment of a first probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof and the second fragment is complementary to the second tag or a fragment thereof, such that when the first tag and the second tag are linked to form a linked nucleic acid, the junction region of the linked nucleic acid is complementary to the contiguous fragment of the first probe.

[0203] In some embodiments, a first fragment of a first probe is complementary to a first tag or a fragment thereof, and another second fragment of a first probe is complementary to a second tag or a fragment thereof; wherein the complementary regions do not include the unconjugated ends of the first and second tags.

[0204] In some embodiments, the first solid surface is coupled to both the first probe and the second probe.

[0205] In some embodiments, (i) the complementary fragment of the first tag and the first probe consists of 10 to 25 base pairs; (ii) the complementary fragment of the second tag and the second probe consists of 10 to 25 base pairs; or both (i) and (ii).

[0206] Also provided herein are assay methods that use one capture binder. In some embodiments, provided herein are assay methods for detecting an analyte in a sample, comprising: (1) mixing a first binder, a second binder, and the sample in solution; wherein the first and second binders bind to non-interfering epitopes on the analyte to form immune complexes; and wherein the immune complexes are captured on a first solid surface in contact with the solution via hybridization between a first nucleic acid tag ("first tag") conjugated to the first binder and a first nucleic acid capture probe ("first probe") coupled to the first solid surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface via dissociation of the hybridization between the first tag and the first probe; (4) introducing a second solid surface coupled with a second nucleic acid probe ("second probe") and recapturing the immune complexes onto the second solid surface via hybridization between the first tag and the second probe; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. An assay method comprising:

[0207] In some embodiments, the second probe is the same as the first probe.

[0208] In some embodiments, step (1) comprises forming an immune complex in solution before capturing the immune complex on a first solid surface. In some embodiments, step (1) comprises pre-capturing a first binder on a first solid surface before capturing the immune complex on the first solid surface. In some embodiments, in step (1), the immune complex is formed in solution and simultaneously captured on the first solid surface.

[0209] In some embodiments, the assay methods provided herein include at least one additional recapture cycle between steps (5) and (6), comprising: releasing the immune complex from the solid surface on which it is captured; recapturing the immune complex on an additional solid surface coupled to (a) the first probe, (b) the second probe, or (c) another nucleic acid probe that hybridizes to the first tag; and washing the additional solid surface to remove unbound molecules.

[0210] In some embodiments, (i) the first probe is directly coupled to a first solid surface; (ii) the second probe is directly coupled to a second solid surface; or both (i) and (ii).

[0211] In some embodiments, (i) the first probe hybridizes to a universal probe that is directly coupled to a first solid surface; (ii) the second probe hybridizes to a universal probe that is directly coupled to a second solid surface; or both (i) and (ii).

[0212] In some embodiments, (i) the first probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to a first solid surface; (ii) the second probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to a second solid surface; or both (i) and (ii).

[0213] In some embodiments, the second binder is conjugated to a second nucleic acid tag ("second tag").

[0214] In some embodiments, the first tag and the second tag are cooperatively captured to a first solid surface in step (1).

[0215] In some embodiments, the first fragment of the first probe is complementary to a first tag or a fragment thereof, and the second fragment of the first probe is complementary to a second tag or a fragment thereof; wherein the complementary regions include the unconjugated ends of the first and second tags.

[0216] In some embodiments, the contiguous fragment of a first probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof and the second fragment is complementary to the second tag or a fragment thereof, such that when the first tag and the second tag are linked to form a linked nucleic acid, the junction region of the linked nucleic acid is complementary to the contiguous fragment of the first probe.

[0217] In some embodiments, a first fragment of a first probe is complementary to a first tag or a fragment thereof, and another second fragment of a first probe is complementary to a second tag or a fragment thereof; wherein the complementary regions do not include the unconjugated ends of the first and second tags.

[0218] In some embodiments, the first solid surface is coupled to both the first probe and an additional nucleic acid probe, wherein the additional probe or a fragment thereof is complementary to the second tag or a fragment thereof.

[0219] In some embodiments, the first tag and the second tag are cooperatively captured to a second solid surface in step (4).

[0220] In some embodiments, the first fragment of the second probe is complementary to a first tag or a fragment thereof, and the second fragment of the second probe is complementary to a second tag or a fragment thereof; wherein the complementary regions include the unconjugated ends of the first and second tags.

[0221] In some embodiments, the contiguous fragment of the second probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first tag and the second tag are linked to form a linked nucleic acid, the junction region of the linked nucleic acid is complementary to the contiguous fragment of the second probe.

[0222] In some embodiments, a first fragment of the second probe is complementary to a first tag or a fragment thereof, and another second fragment of the second probe is complementary to a second tag or a fragment thereof; wherein the complementary regions do not include the unconjugated ends of the first and second tags.

[0223] In some embodiments, the second solid surface is coupled to both the second probe and an additional nucleic acid probe, and the additional probe or a fragment thereof is complementary to the second tag or a fragment thereof.

[0224] In some embodiments, the complementary fragment of the first tag and the first probe consists of 10 to 25 base pairs.

[0225] The assay method provided herein detects an analyte in a sample, which comprises detecting the immune complex in step (6).

[0226] In some embodiments, the sample is a serum sample or a plasma sample.

[0227] In some embodiments, the analyte is a binding pair of two molecules; where a first binder binds to one molecule of the binding pair and a second binder binds to the other molecule of the binding pair.

[0228] In some embodiments, (i) the first binder is an antibody or antibody fragment that specifically binds to the analyte; (ii) the second binder is an antibody or antibody fragment that specifically binds to the analyte, or both (i) and (ii).

[0229] In some embodiments, in step (6): the immune complexes are detected simultaneously as they are captured on the solid surface. In some embodiments, in step (6): the immune complexes are detected after they are released from the solid surface into solution.

[0230] In some embodiments, either the first binder or the second binder is conjugated to a detectable marker. In some embodiments, the first binder is conjugated to a detectable marker. In some embodiments, the second binder is conjugated to a detectable marker. In some embodiments, the detectable marker is a nucleic acid.

[0231] In some embodiments, step (6) comprises linking a first tag and a second tag to generate the nucleic acid reporter, and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second tag.

[0232] In some embodiments, step (6) comprises (a) linking a surrogate nucleic acid of a first tag to a second tag (a "second surrogate") to generate a nucleic acid reporter and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second surrogate; (b) linking a surrogate nucleic acid of a first tag to a second tag (a "first surrogate") to generate a nucleic acid reporter and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second tag; or (c) linking a first surrogate to a second surrogate to generate a nucleic acid reporter and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second surrogate; wherein the first tag or fragment thereof is complementary to the first surrogate or fragment thereof, and the second tag or fragment thereof is complementary to the second surrogate or fragment thereof.

[0233] In some embodiments, the nucleic acid reporter is formed by proximity ligation. In some embodiments, the nucleic acid reporter is formed by proximity extension.

[0234] In some embodiments, the nucleic acid reporter is detected by qPCR, digital PCR, or next generation sequencing (NGS).

[0235] In some embodiments, the nucleic acid reporter is detected by rolling cycle amplification (RCA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), or QuantiGene assay.

[0236] In some embodiments, the nucleic acid reporter contains an analyte-specific identification barcode ("ID") ("target ID") fragment in the first tag or first surrogate thereof, or in the second tag or second surrogate thereof. In some embodiments, the nucleic acid reporter contains a first target ID in the first tag or first surrogate and a second target ID in the second tag or second surrogate.

[0237] In some embodiments, the assay methods provided herein comprise simultaneously detecting at least two analytes in a sample by simultaneously detecting a unique target ID associated with each analyte.

[0238] In some embodiments, the assay methods provided herein comprise, in step (1), proportionally reducing the signal from at least one of the analytes by adding a non-functional binder to the solution, wherein the non-functional binder competes with the first binder for binding to the analyte but is either unconjugated or conjugated to a presenting group that does not bind to the first acceptor group.

[0239] In some embodiments, at least one analyte is a nucleic acid, and the first and second binders of the nucleic acid analyte comprise nucleic acids that are complementary to different fragments of the nucleic acid analyte.

[0240] In some embodiments, in step (6), detecting the analyte comprises simultaneously detecting the first target ID and the second target ID.

[0241] In some embodiments, the analyte is a binding pair of two molecules; where a first binder binds to one molecule of the binding pair and a second binder binds to the other molecule of the binding pair.

[0242] In some embodiments, the nucleic acid reporter formed in each sample contains an ID ("sample ID") that is sample-specific, where the sample ID is (1) inserted between the first tag or its surrogate and the second tag or its surrogate, (2) contained within the first surrogate or the second surrogate, or (3) ligated to the first tag or its surrogate or the second tag or its surrogate.

[0243] In some embodiments, the assay methods provided herein involve simultaneously detecting an analyte in at least two samples by simultaneously detecting a unique sample ID in a nucleic acid reporter associated with each sample.

[0244] In some embodiments, the nucleic acid reporter comprises (a) a target ID in the first tag or first surrogate, or in the second tag or second surrogate, and (b) a sample ID that is (1) inserted between the first tag or surrogate and the second tag or surrogate, (2) contained within the first surrogate or second surrogate, or (3) ligated to the first tag or surrogate, or the second tag or surrogate.

[0245] In some embodiments, the assay methods provided herein comprise simultaneously detecting at least two analytes in at least two samples by simultaneously detecting a unique sample ID and a unique target ID in a nucleic acid reporter associated with each analyte in each sample.

[0246] In some embodiments, the nucleic acid reporter is detected by multiplexed qPCR, multiplexed digital PCR, or NGS.

[0247] In some embodiments, (i) the first solid surface is a magnetic particle surface or a well of a microtiter plate; (ii) the second solid surface is a magnetic particle surface or a well of a microtiter plate; or both (i) and (ii).

[0248] In some embodiments, provided herein is a system for detecting an analyte in a sample, comprising a first binder, a second binder, a first presenting group, a second presenting group, a first accepting group, and a second accepting group; wherein (i) the first and second binders bind to non-interfering epitopes on the analyte, and (ii) the first and second presenting groups bind to the first and second accepting groups, respectively.

[0249] In some embodiments, the systems provided herein further comprise a first solid surface and a second solid surface.

[0250] In some embodiments, (i) the first solid surface is a magnetic particle surface or a well of a microtiter plate; (ii) the second solid surface is a magnetic particle surface or a well of a microtiter plate; or both (i) and (ii).

[0251] In some embodiments, a first solid surface is coupled to a first accepting group and a second first solid surface is coupled to a second accepting group.

[0252] In some embodiments, the systems provided herein further comprise a detectable marker.

[0253] In some embodiments, the detectable marker is conjugated to the first binder or the second binder.

[0254] In some embodiments, a first binder is conjugated to a first presenting group and a second binder is conjugated to a second presenting group.

[0255] In some embodiments, (i) the first presenting group is linked to the first accepting group via a thioester group, a disulfide bond, or a cleavable bond; (ii) the second presenting group is linked to the second accepting group via a thioester group, a disulfide bond, or a cleavable bond; or both (i) and (ii).

[0256] In some embodiments, (i) the first presenting group is attached to the first acceptor group via a photocleavable, chemically cleavable, or enzymatically cleavable bond; (ii) the second presenting group is attached to the second acceptor group via a photocleavable, chemically cleavable, or enzymatically cleavable bond; or both (i) and (ii).

[0257] In some embodiments, provided herein is a system for detecting an analyte in a sample, comprising a first binder, a second binder, a first nucleic acid tag ("first tag"), a second nucleic acid tag ("second tag"), a first nucleic acid capture probe ("first probe"), and a second nucleic acid capture probe ("second probe"); wherein (i) the first and second binders bind to non-interfering epitopes on the analyte; (ii) the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; and (iii) the second probe or a fragment thereof is complementary to the second tag or a fragment thereof.

[0258] In some embodiments, the systems provided herein further comprise a first solid surface and a second solid surface.

[0259] In some embodiments, (i) the first solid surface is a magnetic particle surface or a well of a microtiter plate; (ii) the second solid surface is a magnetic particle surface or a well of a microtiter plate; or both (i) and (ii).

[0260] In some embodiments, a first solid surface is coupled to a first probe and a second first solid surface is coupled to a second probe.

[0261] In some embodiments, the first binder is conjugated to a first tag and the second binder is conjugated to a second tag.

[0262] In some embodiments, (i) the complementary fragment of the first tag and the first probe consists of 10 to 25 base pairs; (ii) the complementary fragment of the second tag and the second probe consists of 10 to 25 base pairs; or both (i) and (ii).

[0263] In some embodiments, (i) the first binder is an antibody or antibody fragment that specifically binds to the analyte; (ii) the second binder is an antibody or antibody fragment that specifically binds to the analyte; or both (i) and (ii).

[0264] Provided herein is a system for detecting an analyte in a sample, comprising a first binder, a second binder, a first nucleic acid tag ("first tag"), a first nucleic acid capture probe ("first probe"), and a second nucleic acid capture probe ("second probe"); wherein (i) the first and second binders bind to non-interfering epitopes on the analyte; (ii) the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; and (iii) the second probe or a fragment thereof is complementary to the first tag or a fragment thereof.

[0265] In some embodiments, the second probe is the same as the first probe.

[0266] In some embodiments, the first binder is conjugated to a first tag.

[0267] In some embodiments, the systems provided herein further comprise a detectable marker. In some embodiments, either the first binder or the second binder is conjugated to a detectable marker.

[0268] In some embodiments, the systems provided herein further comprise a second nucleic acid tag ("second tag"). In some embodiments, the second tag is conjugated to a second binder.

[0269] In some embodiments, the fragment of the first probe is complementary to the second tag or a fragment thereof.

[0270] In some embodiments, the systems provided herein further comprise a first solid surface and a second solid surface.

[0271] In some embodiments, (i) the first solid surface is a magnetic particle surface or a well of a microtiter plate; (ii) the second solid surface is a magnetic particle surface or a well of a microtiter plate; or both (i) and (ii).

[0272] In some embodiments, (1) a first solid surface is coupled to a first probe; (2) a second solid surface is coupled to a second probe; or both (1) and (2).

[0273] In some embodiments, the systems provided herein further comprise an additional nucleic acid probe, wherein the additional probe or a fragment thereof is complementary to the second tag or a fragment thereof.

[0274] In some embodiments, the systems provided herein further comprise a first solid surface and a second solid surface, wherein the first solid surface is coupled to the first probe and the additional probe, and the second solid surface is coupled to the second probe and the additional probe.

[0275] In some embodiments, (i) the first binder is an antibody or antibody fragment that specifically binds to the analyte; (ii) the second binder is an antibody or antibody fragment that specifically binds to the analyte; or both (i) and (ii).

[0276] In some embodiments, (i) the complementary fragment of the first tag and the first probe consists of 10 to 25 base pairs; (ii) the complementary fragment of the second tag and the second probe consists of 10 to 25 base pairs; or both (i) and (ii).

[0277] In some embodiments, the system is included in a kit.

[0278] In some embodiments, the kit further comprises a binding buffer, an immobilization buffer, a wash buffer, a release buffer, or any combination thereof. [Brief explanation of the drawings]

[0279] (4. Brief description of the drawings) [Figure 1] Figure 1 is a schematic diagram of the single molecule array (SIMOA) assay workflow. As shown, this technology is based on a sandwich ELISA, but in the final readout step of the assay, molecules carrying signal-generating moieties are isolated, detected, and counted one at a time. The assay's limit of detection (LOD) is limited to the two- to one-digit fM range, depending on the quality of the antibody pair used (Yeung et al., J. Imm. Meth. 437: 53-63 (2016)).

[0280] [Figure 2] Figure 2 is a schematic diagram of the immuno-PCR workflow. As shown, a nucleic acid segment pre-conjugated to a detection antibody is used as a reporter for the immune complex, and PCR is used to amplify the reporter and generate a detectable signal. Despite the significant enhancement in signal intensity resulting from PCR amplification of the reporter, the improvement in LOD was modest, usually about 10-fold compared to sandwich ELISA (Potuckova, J. Immu. Meth. 371: 38-47 (2011)).

[0281] [Figure 3] Figures 3A-3C are schematic diagrams of proximity ligation assays ("PLA") (Figure 3A), proximity extension assays ("PEA") (Figure 3B), and solid-phase PLA (Figure 3C). As shown, a nucleic acid reporter is generated when two binders are in close proximity so that their attached nucleic acids can be ligated (PLA; Figure 3A) or extended (PEA; Figure 3B). Proximity-based detection assays also have LODs in the mid-to-low fM range. In solid-phase PLA, two binders for a target analyte are each conjugated to a nucleic acid, and a third binder captures the analyte to a solid surface (Figure 3C). Solid-phase proximity assays have demonstrated LODs in the single-digit fM range (Nong RY, Nature Protocols, 8(6): 1234-1249 (2013)). However, the need for three non-interfering antibodies against the same target protein presents a major challenge in assay development.

[0282] [Figure 4] Figures 4A-4D are schematic diagrams of assay methods provided herein that use capture and release mechanisms. Figure 4A shows an assay method involving two capture binders. Figure 4B shows an assay method involving two capture binders, using a nucleic acid tag and a probe for capture and release. Figure 4C shows an assay method involving one capture binder that utilizes renewable binding between a first presenting group and a first accepting group. Figure 4D shows an assay method involving one capture binder that utilizes hybridization between the same nucleic acid tag and probe pair for capture and release.

[0283] [Figure 5]5A-5C are schematic diagrams of assay methods provided herein using various nucleic acid reporter configurations. Nucleic acid reporters are generated by ligating a surrogate of a first tag ("first surrogate") to a second tag (FIG. 5A), by ligating a first tag to a surrogate of a second tag ("second surrogate") (FIG. 5B), or by ligating a first surrogate to a second surrogate (FIG. 5C). While ligation is shown in the figures, reporters can also be generated by extension.

[0284] [Figure 6] Figures 6A-6D are schematic diagrams of exemplary capture configurations used in the assay methods provided herein. Figure 6A shows a direct capture configuration. Figure 6B shows an indirect capture configuration using a universal probe. Figure 6C shows an indirect capture configuration using a biotin / streptavidin pair. Figure 6D shows a direct cooperative capture configuration in which a first nucleic acid tag and a second nucleic acid tag cooperatively bind to a nucleic acid capture probe.

[0285] [Figure 7] 7A-7C are schematic diagrams of exemplary cooperative capture configurations used in the assay methods provided herein. Figure 7A illustrates cooperative capture in which consecutive fragments of a probe hybridize to the junction region of a first tag and a second tag. Figure 7B illustrates cooperative capture in which a fragment of the probe hybridizes to a fragment of the first tag and another fragment of the probe hybridizes to a fragment of the second tag. Figure 7C illustrates cooperative capture in which a solid surface couples to both a first probe and a second probe, which capture the first tag and the second tag, respectively.

[0286] [Figure 8]Figures 8A-8G are schematic diagrams of an embodiment of a NULISA immunoassay. Figures 8A-8B illustrate an assay method in which the capture / release process involves two releasable orthogonal bonds preformed at least once between each binder and its respective solid surface. Figure 8A illustrates detection by formation of a nucleic acid reporter using both nucleic acid tags conjugated to the binder; Figure 8B illustrates detection by iPCR of a nucleic acid molecule conjugated to a second binder. Figures 8C-8D illustrate an assay method involving one releasable and reproducible bond on a first solid surface, in which capture / release is repeatedly preformed between the first binder and multiple solid surfaces and nucleic acid reporters. Figure 8C shows detection by formation of a nucleic acid reporter with both nucleic acid tags conjugated to binders; Figure 8D shows detection by iPCR of a nucleic acid molecule conjugated to a second binder; Figure 8E shows detection of a first target label conjugated to a first binder after capture and release have been performed on both the first binder and the second binder; Figure 8F shows detection of a non-nucleic acid reporter or label; and Figure 8G shows an embodiment in which Binder 1 and Binder 2 indirectly bind to the target, e.g., Binder 1 and Binder 2 bind to a primary antibody that directly binds to the analyte.

[0287] [Figure 9] 9A-9B are schematic diagrams of assay methods provided herein incorporating identification barcodes (IDs) into nucleic acid reporters. In Fig. 9A, one ID is incorporated into one of the nucleic acid tags conjugated to one of the binders. In Fig. 9B, two IDs are incorporated separately into two tags on both binders.

[0288] [Figure 10]10A-10B are schematic diagrams of assay methods provided herein using an indirect ID barcoding approach. In FIG. 10A, a nucleic acid reporter is formed by linking a first nucleic acid surrogate ("first surrogate") that can hybridize to a first tag to a second tag, and an ID is incorporated into the first surrogate. In FIG. 10B, a nucleic acid reporter is formed by linking a first surrogate to a second nucleic acid surrogate ("second surrogate") that can hybridize to a second tag; each surrogate is incorporated with an ID.

[0289] [Figure 11] FIG. 11 is a schematic representation of the multiplexing of the assay methods provided herein, which allows for simultaneous detection of multiple analytes by detecting the unique ID incorporated into a tag conjugated to the binder of each analyte.

[0290] [Figure 12] 12 is a schematic diagram of the assay method provided herein that uses unique IDs to achieve enhanced specificity. As shown, by requiring simultaneous detection of ID 1 and ID 2, which are incorporated into tags conjugated to Binder 1 and Binder 2, respectively, as detection of a "true signal," the method reduces false positive signals associated with detection of only ID 1 or ID 2, but not both.

[0291] [Figure 13] FIG. 13 is a schematic diagram of an assay method provided herein for detecting protein-protein interactions, in which each protein of a binding pair is captured by a binder associated with a unique ID, and the interaction of two proteins is reflected by the simultaneous detection of both IDs in a single immune complex.

[0292] [Figure 14]14A-14C are schematic diagrams of assay methods provided herein that incorporate a sample-specific identifier ("sample ID") and are capable of simultaneously detecting multiple samples. FIG. 14A illustrates the incorporation of a sample ID between a first nucleic acid tag and a second nucleic acid tag in forming a reporter for detection. FIG. 14B illustrates the incorporation of a sample ID in either a first surrogate nucleic acid or a second surrogate nucleic acid that is part of a reporter for detection. FIG. 14C illustrates the ligation of a sample ID to a first nucleic acid tag or a second nucleic acid tag, or surrogates thereof, in forming a reporter for detection.

[0293] [Figure 15] 15A-15J are schematic diagrams of the assay methods provided herein for detecting nucleic acid analytes, including the formation of analyte-binder complexes in solution (FIG. 15A), capture of the immune complexes on a first solid surface (FIG. 15B), release of the immune complexes from the first surface (FIG. 15C), recapture of the immune complexes on a second surface (FIG. 15D), and generation of a reporter (FIG. 15E); and FIGS. 15F-15J show another workflow for nucleic acid detection, including the formation of analyte-binder complexes in solution (FIG. 15F), capture of the analyte-binder complexes on a first surface (FIG. 15G), release of the analyte-binder complexes (FIG. 15H), recapture of the analyte-binder complexes on a second surface (FIG. 15I), and generation of a reporter (FIG. 15J).

[0294] [Figure 16]Figure 16 is a schematic diagram of an exemplary NULISA immunoassay configuration. As shown, two binders of an analyte are each conjugated to a nucleic acid tag ("CP" and "L," respectively), where CP is indirectly coupled to a first solid surface (paramagnetic bead 1) via a universal probe (polyT), and L is indirectly coupled to a second solid surface (paramagnetic bead 2) via a streptavidin / biotin linkage. After immunoassay formation and reduction of nonspecific binding via the capture / release mechanism disclosed herein, a nucleic acid reporter of the immune complex is formed by ligation of L to a surrogate R of the CP (which hybridizes to a portion of the CP) and a short oligo SI serving as the sample ID. The target ID (TI) is incorporated as a segment of L. The connector (CNT) is a bridging probe positioned for ligation.

[0295] [Figure 17] FIG. 17 is a titration curve for human EGFR-Fc protein detection using the NULISA immunoassay.

[0296] [Figure 18] 18A-18C show the basic configuration of the nucleic acid-binding immunoconjugates provided herein.

[0297] [Figure 19] 19A-19D show some additional configurations of the nucleic acid-binding immunoconjugates provided herein.

[0298] [Figure 20]Figures 20A-20E show another workflow of NULISA, including the formation of immune complexes in solution (Figure 20A), capture of binder 1 to a first solid surface via a first nucleic acid capture probe molecule ("CP1" or "first probe") (Figure 20B), release of the immune complex from the first surface (Figure 20C), and generation of a nucleic acid reporter (Figure 20D), as well as a comparison of the signal generated when the immune complex is released into solution (right bar of each pair) or not (left bar of each pair) (Figure 20E).

[0299] [Figure 21] Figures 21A-21I show the steps of multiplex NULISA, including incubation (Figure 21A), capture of immune complexes to a first solid surface (Figure 21B), a first wash (Figure 21C), release of immune complexes from the first solid surface (Figure 21D), capture of immune complexes to a second solid surface (Figure 21E), a second wash (Figure 21F), binding of sample labels and ligation to generate nucleic acid reporters containing two analyte-specific identification barcodes ("target IDs") and one sample-specific identification barcode ("sample ID") (Figure 21G), a final wash and elution (Figure 21H), and PCR amplification and detection (Figure 21I). Alternatively, ligation products bearing target IDs and sample IDs can be pooled for sequencing with or without pre-amplification.

[0300] [Figure 22]22 shows an exemplary configuration for NULISAseq. In this configuration, CP can be a first presentation group (first tag), poly-T coupled to a paramagnetic bead can be a first acceptor group (first probe), R can be a first target label containing a first target ID (TI), L can be a second target label containing a second target ID (TI), biotinylated CP2 can be a second presentation group, streptavidin can be a second acceptor group, CNT (short for "connector") can be a sample label having a 3' overhang that binds to the second target label and a 5' overhang that binds to the first target label, SI can be a sample ID within the sample label (CNT), paramagnetic bead 1 can be a first solid surface, and paramagnetic bead 2 can be a second solid surface.

[0301] [Figure 23] Figures 23A-23B show the high sensitivity and multiplexing mechanism of NULISAseq, including (Figure 23A) an exemplary sequence of the "L" fragment of Figure 22, a bridge probe for ligation ("CNT" in Figure 22), and an exemplary sequence of the "R" fragment of Figure 22; and (Figure 23B) an exemplary ligation product for multiplexing NULISAseq with three analytes and three samples.

[0302] [Figure 24] Figures 24A-24B show qPCR quantification and sequencing of analyte P24 (Figure 24A), including barcode assignments and qPCR results across various concentrations of P24, and a comparison of quantification by sequencing and qPCR (Figure 24B).

[0303] [Figure 25] Figures 25A-25C show the results of 5-plex NILISAseq, including the sequencing results for each of the five replicate assays (Figure 25A), the average coefficient of variation (CV) (Figure 25B), and the limit of detection (LOD) for each assay (Figure 25C). DETAILED DESCRIPTION OF THE INVENTION

[0304] (5. Detailed Description) Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described herein, and that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0305] (5.1 Definition) Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, the nomenclature used in connection with, and techniques related to, molecular biology, immunology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

[0306] As used herein, the term "detecting" or its grammatical equivalents is used broadly to include any means of determining the presence (i.e., whether it is present) of an analyte or any form of measurement of an analyte. Thus, "detecting" can include determining, measuring, or assessing the presence or absence, amount, or location of an analyte. Quantitative, semi-quantitative, and qualitative determinations, measurements, or assessments are included. Such determinations, measurements, or assessments can be relative, for example, when two or more different analytes in a sample are being detected, or can be absolute. Thus, the term "quantifying" when used in the context of quantifying a target analyte in a sample can refer to absolute quantification or relative quantification. Absolute quantification can be achieved by inclusion of one or more control analytes of known concentrations and / or by referencing the detection level of the target analyte with known control analytes (e.g., by generating a standard curve). Alternatively, relative quantification can be achieved by comparing the detection levels or amounts between two or more different target analytes to provide a relative quantification of each of the two or more different analytes, i.e., relative to each other.

[0307] As used herein, the term "analyte" can refer to any substance (e.g., molecule) or entity to be detected by the assay methods provided herein. An analyte is the target of the assay methods provided herein. Thus, an analyte can be any biological molecule or chemical compound that needs to be detected, such as a peptide or protein, a nucleic acid molecule, or a small molecule, including organic and inorganic molecules. An analyte can be a cell or a microorganism, including a virus, or a fragment or product thereof. An analyte can be any substance or entity for which a specific binder can be developed and which can simultaneously bind to at least two "binders." In some embodiments, an analyte is a protein or polypeptide. Thus, analytes of interest include proteinaceous molecules such as polypeptides, proteins, or prions, or any molecule containing a protein or polypeptide component, or a fragment thereof. In some embodiments, an analyte is a fully or partially proteinaceous molecule. An analyte can also be a single molecule or a complex containing two or more molecular subunits, which may or may not be covalently bound to each other and which may be the same or different. Therefore, the analyte that can be detected by the assay method described herein can be a complex analyte, which can be a protein complex. Therefore, such a complex can be a homomultimer or a heteromultimer. Molecular aggregates (e.g., proteins) can also be target analytes. Aggregated analytes can be aggregates of the same protein or different proteins. The analyte can also be a complex composed of proteins or peptides or nucleic acid molecules such as DNA or RNA. In some embodiments, the analyte is a complex composed of both proteins and nucleic acids, for example, a regulatory factor such as a transcription factor.

[0308] As used herein, the term "sample" can refer to any biological and clinical sample, including, for example, any cell or tissue sample from an organism, or any body fluid or preparation derived from an organism, and samples such as cell cultures, cell preparations, cell lysates, etc. Environmental samples, such as soil and water samples or food samples, are also included. Samples can be freshly prepared or previously processed in any convenient way (e.g., for storage).

[0309] Representative samples, therefore, include any material containing a biomolecule or any other desired or target analyte, including, for example, food and related products, clinical and environmental samples. Samples can be biological samples, including viruses or cellular material, including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasma, protoplasts, and organelles. Such biological materials include all types of mammalian and non-mammalian cells, plant cells, algae, including blue-green algae, fungi, bacteria, protozoa, and the like. Representative samples also include whole blood and blood-derived products, such as plasma, serum, and buffy coat, blood cells, urine, feces, cerebrospinal fluid, or any other bodily fluid (e.g., respiratory secretions, saliva, milk, etc.), tissue, biopsy, cell culture, cell suspension, conditioned medium, or other samples of cell culture components. Samples can be pre-processed in any convenient or desired manner to prepare them for use in the methods disclosed herein. For example, the sample can be processed by cell lysis or purification, isolation of analytes, and the like.

[0310] As used herein, the term "binding" or its grammatical equivalents refers to an interaction between molecules (e.g., a binder and an analyte, or a presenting group and an accepting group) to form a complex. The interaction can be a non-covalent interaction, including, for example, hydrogen bonding, ionic bonding, hydrophobic interactions, and / or van der Waals interactions. A "binder," as used herein with respect to an analyte, is any molecule or entity capable of binding to the analyte. In some embodiments, a binder specifically binds to its target analyte, i.e., the binder binds to the target analyte with a higher affinity than to other components in the sample. In some embodiments, the binding of a binder to a target analyte is distinguishable from the binding of a binder to non-target analytes in that the binder either does not bind to non-target analytes or binds to them to a negligible or undetectable extent, or any such non-specific binding, if any, is at a relatively low level that can be distinguished. The binding between a target analyte and its binder is typically non-covalent. The binders used in the methods provided herein can be covalently conjugated to a presenting group (e.g., a nucleic acid tag) without substantially eliminating the binding affinity of the binder for its target analyte.

[0311] The binder can be selected to have a high binding affinity for the target analyte. In some embodiments, the binder has a binding affinity of at least about 10 -4 M, at least about 10 -6 M, or at least 10 -9 M or higher for the target analyte. Binders can be a variety of different types of molecules, so long as they exhibit the requisite binding affinity for the target analyte. In other embodiments, binders have moderate or low, e.g., about 10, binding affinity for the target analyte. -4 It may have an affinity of less than M.

[0312] The binder can be a macromolecule. In some embodiments, the binder is an antibody or a binding fragment, derivative, or mimetic thereof. When the binder is an antibody, it can be obtained from a polyclonal composition in which a heterogeneous antibody population with different specificities is conjugated to the same presenting group, or a monoclonal composition in which a homogeneous, identical antibody population with the same specificity to the target analyte is conjugated to the same presenting group. Thus, the binder can be either a monoclonal antibody or a polyclonal antibody.

[0313] In some embodiments, the binder is an antibody fragment, derivative, or mimetic thereof, where the fragment, derivative, or mimetic has the requisite binding affinity for the target analyte. Such antibody fragments or derivatives typically have at least the V of the subject antibody in order to retain the binding characteristics of the subject antibody. H and V L In some embodiments, the binder is an antibody fragment that binds to the analyte. As used herein, an antibody fragment refers to a molecule other than an intact antibody that contains a portion of an antibody and typically an antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), Fv, single-chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), diabodies, triabodies, tetrabodies, minibodies, dual variable domain antibodies (DVDs), single variable domain antibodies (e.g., camelid antibodies, alpaca antibodies), single variable domains of heavy chain antibodies (VHHs), and multispecific antibodies formed from antibody fragments. In some embodiments, the binder is a Fab. In some embodiments, the binder is an scFv. In some embodiments, the binder is a single variable domain antibody.

[0314] In some embodiments, the binder is an antibody mimetic. Antibody mimics can be molecules that, like antibodies, can specifically bind to an antigen but are not structurally related to antibodies. Antibody mimics are typically artificial peptides with a molar mass of approximately 2-20 kDa. Nucleic acids and small molecules may also be considered antibody mimics. Antibody mimics known in the art include affibodies, affilins, affimers, affitins, alphabodies, anticalins, aptamers, avimers, DARPins, Fynomers, Kunitz domain peptides, monobodies, and nanoCLAMPs.

[0315] In some embodiments, suitable for use as binder is polynucleic acid aptamer.Polynucleic acid aptamer can be RNA oligonucleotide, which can selectively bind to protein in roughly the same manner as receptor or antibody (Conrad et al., Methods Enzymol. (1996), 267 (Combinatorial Chemistry), 336-367).Above-mentioned antibody, its fragment, derivative and mimic can be obtained from commercial sources, and / or can be prepared by any convenient technique, if the method of producing polyclonal antibody, monoclonal antibody, its fragment, derivative and mimic, including its recombinant derivative, is known to those skilled in the art (for example, U.S. Patent No. 5,851,829 and 5,965,371).

[0316] In addition to antibody-based peptide / polypeptide or protein-based binding domains, binders can also be lectins, soluble cell surface receptors or derivatives thereof, affibodies, or any combinatorially derived protein or peptide from phage display or ribosome display or any type of combinatorial peptide or protein library.

[0317] The binder can also be a ligand. Ligand binders can have a variety of sizes. In some embodiments, the ligand binder has a size of about 50 to about 10,000 daltons, about 50 to about 5,000 daltons, or about 100 to about 1,000 daltons. In some embodiments, the ligand binder has a size with a molecular weight of about 10,000 daltons or greater.

[0318] In some embodiments, the binder is a small molecule that can bind to the target analyte with the requisite affinity. The small molecule is a small organic molecule. The small molecule can contain one or more functional groups necessary for structural interaction with the target analyte, such as groups necessary for hydrophobic interaction, hydrophilic interaction, electrostatic interaction, or covalent interaction. When the target analyte is a protein, the small molecule binder contains functional groups necessary for structural interaction with the protein, such as hydrogen bonding, hydrophobic-hydrophobic interaction, electrostatic interaction, etc., and usually contains at least amine, amide, sulfhydryl, carbonyl, hydroxyl, or carboxyl groups. In some embodiments, at least two of the functional groups are included. The small molecule binder can also contain a region that can be modified and / or participate in covalent bonding with a presentation group (e.g., a nucleic acid tag) without substantially adversely affecting the ability of the small molecule to bind to its target analyte.

[0319] Small molecule binders can also include cyclic carbon or heterocyclic structures and / or aromatic or aromatic polycyclic structures substituted with one or more of the above functional groups. Small molecule binders can also contain structures found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Such compounds can be screened to identify compounds of interest. A variety of different screening protocols are known in the art.

[0320] Small molecule binders can also be derived from natural or synthetic compounds, which can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or can be readily produced. Furthermore, natural or synthetically produced libraries and compounds can be easily modified by conventional chemical, physical, and biochemical means and used to produce combinatorial libraries. Known small molecules can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogs. Thus, small molecule binders can be obtained from libraries of natural or synthetic molecules, including libraries of compounds produced by combinatorial means, i.e., compound diversity combinatorial libraries. When obtained from such libraries, small molecule binders are selected for exhibiting some desirable affinity for a protein target in a convenient binding affinity assay.

[0321] The assay methods provided herein use a first binder and a second binder that bind to a non-interfering "epitope" of the analyte. An epitope of an analyte, as understood in the art, refers to a site on the surface of the analyte to which a binder binds. An epitope can be a localized region on the surface of the analyte. An epitope can consist of a chemically active surface grouping of molecules, such as amino acids or sugar side chains. An epitope can have specific three-dimensional structural and specific charge characteristics. An epitope can be a contiguous fragment of the analyte molecule. An epitope can also be a molecule to which multiple non-contiguous fragments of an antigen bind. When the analyte is a polypeptide or protein, the epitope can comprise a contiguous or non-contiguous sequence along the primary sequence of the polypeptide chain. In some embodiments, the first binder and second binder used in the assay methods disclosed herein are of the same molecular type. For example, both the first binder and the second binder can be monoclonal antibodies that bind to a non-interfering epitope of the analyte. In some embodiments, the first binder and the second binder can be different, for example, the first binder can be an antibody and the second binder can be a small molecule.

[0322] The term "binding moiety" when used with respect to an analyte refers to a moiety comprising a molecule or group of molecules such that the moiety as a whole is capable of specifically binding to the analyte. A binding moiety can comprise one or more binders, one or more target labels, one or more sample labels, and / or one or more presentation groups. A binding moiety can also comprise a binder, a target label, a sample label, and / or a presentation group. Alternatively, a binding moiety can comprise a binder, a target label, and / or a presentation group. Molecules in a binding moiety can be bound as a moiety through covalent intermolecular interactions, noncovalent intermolecular interactions, or a combination of covalent and noncovalent intermolecular interactions. Alternatively, molecules in a binding moiety can be bound through interactions with molecules that are not part of the binding moiety, such as the analyte or one or more receptor groups. Furthermore, molecules in a binding moiety can be bound as a moiety (i) through intermolecular interactions between molecules within the binding moiety and (ii) through interactions with molecules that are not part of the binding moiety, such as the analyte or one or more receptor groups. In one embodiment, a binding moiety comprises or consists of a binder. In some embodiments, the binding moiety comprises or consists of a target label. In certain embodiments, the binding moiety comprises or consists of a presentation group. In other embodiments, the binding moiety comprises or consists of a sample label. In one embodiment, the binding moiety comprises or consists of a binder and a target label. In some embodiments, the binding moiety comprises or consists of a binder and a presentation group. In certain embodiments, the binding moiety comprises or consists of a binder and a sample label. In further embodiments, the binding moiety comprises or consists of a target label and a presentation group. In one embodiment, the binding moiety comprises or consists of a target label and a sample label. In other embodiments, the binding moiety comprises or consists of a presentation group and a sample label. In yet other embodiments, the binding moiety comprises or consists of a binder, a target label, and a presentation group. In some embodiments, the binding moiety comprises or consists of a binder, a target label, and a sample label.In certain embodiments, the binding moiety comprises or consists of a binder, a presenting group, and a sample label. In some embodiments, the binding moiety comprises or consists of a target label, a presenting group, and a sample label. In other embodiments, the binding moiety comprises or consists of a binder, a target label, a presenting group, and a sample label. In some embodiments, the binding moiety comprises or consists of any one of a binder, a target label, a presenting group, and a sample label. In some embodiments, the binding moiety comprises or consists of any two of a binder, a target label, a presenting group, and a sample label, in any combination or permutation. In some embodiments, the binding moiety comprises or consists of any three of a binder, a target label, a presenting group, and a sample label, in any combination or permutation. In some embodiments, the binding moiety comprises or consists of all four of a binder, a target label, a presenting group, and a sample label.

[0323] The terms "presenting group" and "accepting group" are used in conjunction with each other herein and refer to a binding pair that can form a complex under appropriate conditions. When used in the assay methods disclosed herein, the presenting group can be conjugated to a binder of the target analyte, and the accepting group can be coupled to a solid surface. Thus, the bond between the presenting group and the accepting group allows the analyte to be captured on the solid surface. In some embodiments, the bond formed between the presenting group and the accepting group is "releasable," allowing the captured binder to be released from the solid surface. In some embodiments, the bond formed between the presenting group and the accepting group is "regenerable," allowing the binder to be recaptured on another solid surface coupled to the same accepting group. As with the binding pair between a "binder" and its "analyte" discussed above, the binding pair between a "presenting group" and an "accepting group" can take a variety of forms. Examples of binding pairs between a "presenting group" and an "acceptor group" include, but are not limited to, an antigen and an antibody to the antigen (including fragments, derivatives, or mimetics thereof), a ligand and its receptor, complementary strands of nucleic acid, biotin and avidin (or streptavidin or neutravidin), a lectin and a carbohydrate (or vice versa). Further binding pairs between a "presenting group" and an "acceptor group" include fluorescein and anti-fluorescein, digoxigenin / anti-digoxigenin, and DNP (dinitrophenol) / anti-DNP (or vice versa). In some embodiments, the binding pair between a "presenting group" and an "acceptor group" is a complementary strand of nucleic acid, referred to as a "tag" and a "probe." In some embodiments, the binding pair between a "presenting group" and an "acceptor group" is an antigen and an antibody, or an antigen and an antibody fragment.

[0324] The term "target label" refers to a moiety that facilitates detection and identification of a target molecule. The term "sample label" refers to a moiety that facilitates detection and identification of a sample source of a target. Suitable labels for target and sample labels include labels that can provide an identifier that can be associated with a particular target or sample. A common label that can be used for target and / or sample labeling in the context of the present disclosure is a sequence of nucleotides that can be associated with a target or sample via sequencing. In some embodiments, the target label comprises a target ID. In certain embodiments, the target label consists of a target ID. In other embodiments, the target label is a target ID. In some embodiments, the sample label comprises a sample ID. In certain embodiments, the sample label consists of a sample ID. In other embodiments, the sample label is a sample ID. Additional labels suitable for target and sample labels of the present disclosure include molecules that contain other distinguishable or correlative information, such as a fluorescent molecule or combination or sequence of fluorescent molecules, and / or a colorimetric moiety or combination or sequence of colorimetric moieties. Other labels contemplated for the present disclosure include luminescent, light-scattering, radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, and the like. Patents that teach the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Many labels are commercially available and can be used in connection with the present invention.

[0325] The term "identification barcode" or "ID," when used in reference to a target or sample, refers to a molecule or series of molecules that can be used to directly or indirectly identify a target or sample by virtue of identifying information contained in the molecule or series of molecules. Such an ID can be a nucleic acid molecule having a given sequence, a unique fluorescent label, a unique colorimetric label, a sequence of fluorescent labels, a sequence of colorimetric labels, or any other molecule or combination of molecules, so long as the molecule or combination of molecules used as the ID identifies or otherwise distinguishes a particular target or sample from other targets or samples and is capable of being associated with the intended target or sample. Nucleic acid molecules used as such IDs are also known as barcode sequences. Such IDs can also be further derived molecules containing information that is derived from, but not identical to, the original ID, so long as such derived molecule or derived information identifies or otherwise distinguishes a particular target or sample from other targets or samples and is capable of being associated with the intended target or sample. For example, a nucleic acid ID can include both the original nucleic acid barcode sequence and / or the reverse complement of the original nucleic acid barcode sequence, since both can distinguish and associate with an intended target or sample. The barcode sequence can be any sequence, natural or non-natural, that is not present in the intended sample, the intended target, or any portion of the intended sample or target unless introduced as a barcode sequence, such that the barcode sequence can distinguish and associate with the sample or target. The barcode sequence can be unique to a single nucleic acid species within a population, or the barcode sequence can be shared by several different nucleic acid species within a population. Each nucleic acid probe within a population can include a barcode sequence that is different from all other nucleic acid probes within the population. Alternatively, each nucleic acid probe within a population can include a barcode sequence that is different from some or most other nucleic acid probes within the population. As a specific example, all reporters generated from immune complexes derived from a single sample can have the same sample barcode sequence (sample ID).As another example, all reporters generated from immune complexes derived from the same sample can have different target barcode sequences (target IDs).Furthermore, all reporters generated from immune complexes derived from the same sample, against the same target, and with the same binder can have the same target barcode sequence (target ID).

[0326] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (only); and B (only). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (only); B (only); and C (only).

[0327] 5.2 Immunoassays Provided herein is an assay method that addresses the limitations of existing immunoassays and enables single molecule detection of immune complexes through nucleic acid-based signal amplification. Provided herein is a method that reduces background signals through a capture and release mechanism. Thus, provided herein is an assay method for detecting an analyte in a sample, comprising a capture and release mechanism. In some embodiments, the capture and release mechanism is based on the hybridization and dissociation of nucleic acid pairs.

[0328] 5.2.1 Capture and Release Mechanisms The assay methods provided herein use a capture and release mechanism to reduce nonspecific background signals. The process of capturing immune complexes on a solid surface and releasing them back into solution can be applied to various assay formats disclosed herein (e.g., Figures 4A-4D and 8A-8D).

[0329] 5.2.1.1 Capture and Release Using Two Capture Binders In some embodiments, as shown in Figures 4A, 8A, and 8B, the assay methods provided herein use a capture and release mechanism involving two binders that can be captured by two acceptor groups on two solid surfaces, respectively. In some embodiments, provided herein are assay methods for detecting an analyte in a sample, comprising the following steps: (1) mixing a first binder, a second binder, and the sample in solution, wherein the first and second binders bind to non-interfering epitopes on the analyte and form immune complexes, which are captured on the first solid surface in contact with the solution via binding between a first presenting group conjugated to the first binder and a first accepting group coupled to a first surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (4) introducing a second solid surface and recapturing the immune complex via binding between a second presenting group conjugated to the second binder and a second accepting group coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. The assay method includes:

[0330] In Figures 4A, 8A, and 8B, capture / release of a first binder ("Binder 1") to / from a first solid surface ("Surface 1") and capture / release of a second binder ("Binder 2") to / from a second solid surface ("Surface 2") are achieved by two bonds between a presenting group ("PG") and an accepting group ("RG") that are bioorthogonal (i.e., each independent and specific). The bond between the first presenting group ("PG1") and the first accepting group ("RG1"), i.e., the first bond ("Bond 1"), is releasable. In some embodiments, the bond between the second presenting group ("PG2") and the second accepting group ("RG2"), i.e., the second bond ("Bond 2"), is also releasable, and immune complexes can be detected either on Surface 2 or after release from Surface 2. In some embodiments, Bond 2 is not releasable, and immune complexes can be detected on Surface 2.

[0331] As one skilled in the art will appreciate, additional rounds of capture / release will further reduce nonspecific background signals. In some embodiments, bond 1 is renewable, and at least one additional round of capture / release can be performed via binder 1. Specifically, immune complexes released from surface 2 can be recaptured by new surface 1 by forming another bond between PG1 on binder 1 and RG1 on new surface 1. In some embodiments, bond 2 is renewable, and at least one additional round of capture / release can be performed via binder 2. Specifically, immune complexes released from either surface 1 or surface 2 can be recaptured by new surface 2 by forming another bond between PG2 on binder 2 and RG2 on new surface 2. In some embodiments, both bond 1 and bond 2 are renewable, and multiple recapture cycles can be performed via bond 1, bond 2, or both. In some embodiments, neither bond 1 nor bond 2 is renewable, and only one capture / release cycle is performed.

[0332] Thus, in some embodiments, the assay methods provided herein further comprise at least one additional recapture cycle between steps (5) and (6), comprising: releasing the immune complex from the solid surface on which it is captured, recapturing the immune complex onto a further solid surface coupled to a first acceptor group, and washing the further solid surface to remove unbound molecules. In some embodiments, the assay methods provided herein further comprise at least one additional recapture cycle between steps (5) and (6), comprising: releasing the immune complex from the solid surface on which it is captured, recapturing the immune complex onto a further solid surface coupled to a second acceptor group, and washing the further solid surface to remove unbound molecules. Additional recapture cycles can be included.

[0333] The releasable bond can be achieved by many different approaches known to those skilled in the art of protein immobilization. For example, in some embodiments, the releasable bond is attached via a thioester group (e.g., U.S. Patent No. 4,284,553). In some embodiments, the releasable bond is a cleavable bond (e.g., Leriche, Bioorganic & Med. Chem. 20(2): 571-581(2012)). In some embodiments, the releasable bond is a disulfide bond (e.g., Chan, Biochemistry 15(19): 4215-4222(1976)). In some embodiments, the releasable bond is a photocleavable bond (e.g., photocleavable spacers available from Integrated DNA Technologies; Wan, PLoS ONE 13(2): e0191987(2018)). In some embodiments, the releasable bond is a bond that can be cleaved by an appropriate enzymatic activity, including, for example, phosphodiester, phospholipid, ester, or b-galactose. In some embodiments, the releasable bond is a bond that can be cleaved by a chemoenzymatic reaction, such as the Staphy-eSrtA pair (e.g., Ham et al., Nature Communications 7:11140 (2016)) and others (Rabuka, Curr. Opin. Chem. Biol. 14, 790-796 (2010); Rashidian, J. Am. Chem. Soc. 134:8455-8467 (2012)); Kosa, Nat. Methods 9, 981-984 (2012)). In some embodiments, the releasable bond is formed between an arginine residue and an adsorbent derivatized with 4-(oxoacetyl)phenoxyacetic acid (e.g., Duerksen-Hughes, Biochemistry, 28(21):8530-6(1989)). In some embodiments, the releasable bond is a non-covalent bond that is broken by binding competition (e.g., Nguyen, Biomol. Eng. 22(2005) 147-150).Renewable bonds can also be achieved by many different approaches known to those skilled in the art of protein immobilization. For example, non-covalent bonds, including hydrogen bonds formed between binding pairs (e.g., antigen and antibody, ligand and receptor, complementary nucleic acids, etc.), can be renewable. Releasable and renewable bonds can also be achieved, for example, by using metal affinity (e.g., Cheung, Appl. Microbiol. Biotechnol. 96, 1411-1420 (2012)), N-halamine structures (e.g., Hui, Biomacromolecules 14 585-601 (2013)), or disulfide bonds (e.g., Boitieux, Anal. Chim. Acta 197: 229-237 (1987)).

[0334] In some embodiments, in step (3), the immune complex is liberated from the first solid surface through binding competition by adding an excess amount of either a free first presenting group or a free first accepting group to the solution. A "free" presenting group refers to a presenting group that is not conjugated to a binder. A "free" accepting group refers to an accepting group that is not coupled to a solid surface.

[0335] In some embodiments, the releasable and reproducible bond is formed through nucleic acid hybridization, where the presenting group and the accepting group are complementary nucleic acids. In some embodiments, the presenting group is a nucleic acid that can bind to an accepting group that is a DNA / RNA-specific protein or aptamer binding partner (e.g., US5312730). In some embodiments, the accepting group is a nucleic acid that can bind to a presenting group that is a DNA / RNA-specific protein or aptamer binding partner.

[0336] In some embodiments, the first presenting group is a first nucleic acid tag ("first tag") and the first accepting group is a first nucleic acid capture probe ("first probe"), where the first probe or a fragment thereof is complementary to the first tag or a fragment thereof. In some embodiments, the second presenting group is a second nucleic acid tag ("second tag") and the second accepting group is a second nucleic acid capture probe ("second probe"), where the second probe or a fragment thereof is complementary to the second tag or a fragment thereof.

[0337] In some embodiments, as shown in Figure 4B, the first presenting group is a first tag, the first accepting group is a first probe, and the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; and the second presenting group is a second tag, and the second accepting group is a second probe, and the second probe or a fragment thereof is complementary to the second tag or a fragment thereof. Thus, provided herein is an assay method for detecting an analyte in a sample, comprising the following steps: (1) mixing a first binder, a second binder, and the sample in solution, wherein the first and second binders bind to non-interfering epitopes on the analyte and form immune complexes, and wherein the immune complexes are captured on a first solid surface in contact with the solution via hybridization between a first tag conjugated to the first binder and a first probe coupled to the first surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface via dissociation of the hybridization between the first tag and the first probe; (4) introducing a second solid surface and recapturing the immune complexes onto the second solid surface via hybridization between a second tag conjugated to the second binder and a second probe coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. The assay method includes:

[0338] In some embodiments, the assay methods provided herein further comprise at least one additional recapture cycle between steps (5) and (6), comprising: releasing the immune complex from the solid surface on which it is captured via dissociation of hybridization between the second tag and the second probe, recapturing the immune complex on a further solid surface coupled to a first acceptor group, and washing the further solid surface to remove unbound molecules. In some embodiments, the assay methods provided herein further comprise at least one additional recapture cycle between steps (5) and (6), comprising: releasing the immune complex from the solid surface on which it is captured via dissociation of hybridization between the first tag and the first probe, recapturing the immune complex on a further solid surface coupled to a second acceptor group, and washing the further solid surface to remove unbound molecules.

[0339] 5.2.1.2 Capture and Release Using One Capture Binder In some embodiments, as shown in Figures 4C, 8C, and 8D, the assay methods provided herein use a capture and release mechanism in which two binders are used to form an immune complex, but only one binder is captured by a solid surface via one releasable and renewable bond. In some embodiments, the first binder is captured sequentially by two solid surfaces. In some embodiments, provided herein is an assay method for detecting an analyte in a sample, comprising the following steps: (1) mixing a first binder, a second binder, and the sample in solution; wherein the first and second binders bind to non-interfering epitopes on the analyte to form immune complexes; and wherein the immune complexes are captured on a first solid surface in contact with the solution via binding between a first presenting group conjugated to the first binder and a first accepting group coupled to the first solid surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (4) introducing the immune complex to a second solid surface and recapturing the immune complex via binding between the first presenting group and a second accepting group coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. The assay method includes:

[0340] The exact same capture / release mechanism can be repeated on a second solid surface. Thus, in some embodiments, the second accepting group is the same as the first accepting group.

[0341] As shown in Figures 4C, 8C, and 8D, capture / release can be achieved through a releasable and reproducible bond between a first presenting group ("PG1") of a first binder ("Binder 1") and a first accepting group ("RG1") of a first solid surface ("Surface 1"). The same presenting group (PG1) can then form a second bond ("Bond 2") with a second accepting group ("RG2") of a second solid surface ("Surface 2"). RG2 can be the same as RG1. In some embodiments, Bond 2 is also releasable, and immune complexes can be detected either on Surface 2 or after release from Surface 2. In some embodiments, Bond 2 is not releasable, and immune complexes can be detected on Surface 2.

[0342] As one skilled in the art will appreciate, additional rounds of capture / release will further reduce nonspecific background signals. Because bond 1 is renewable, at least one additional round of capture / release can be performed via binder 1. Specifically, immune complexes released from surface 2 can be recaptured by new surface 1 by forming another bond between PG1 on binder 1 and RG1 on new surface 1. In some embodiments, bond 2 is also renewable, and immune complexes released from surface 1 or surface 2 can be recaptured by new surface 2 by forming another bond between PG1 on binder 1 and RG2 on new surface 2. In some embodiments, both bond 1 and bond 2 are renewable, and multiple recapture cycles can be performed via bond 1, bond 2, or both.

[0343] Thus, in some embodiments, the assay methods provided herein further comprise at least one additional recapture cycle between steps (5) and (6), comprising: releasing the immune complex from the solid surface on which it is captured, recapturing the immune complex onto a further solid surface coupled with a first acceptor group, and washing the further solid surface to remove unbound molecules. In some embodiments, the assay methods provided herein further comprise at least one additional recapture cycle between steps (5) and (6), comprising: releasing the immune complex from the solid surface on which it is captured, recapturing the immune complex onto a further solid surface coupled with a second acceptor group, and washing the further solid surface to remove unbound molecules. Additional recapture cycles can be included.

[0344] As noted above, releasable and renewable bonds can be achieved by many different approaches known to those skilled in the art of protein immobilization, including those disclosed herein. In some embodiments, releasable and renewable bonds are formed via nucleic acid hybridization, where the presenting group and the accepting group comprise nucleic acids that are complementary to each other.

[0345] In some embodiments, the first presenting group is a nucleic acid tag ("first tag") and the first accepting group is a nucleic acid capture probe ("first probe"), wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof. Thus, as shown in Figure 4D, provided herein is an assay method for detecting an analyte in a sample, comprising: (1) mixing a first binder, a second binder, and the sample in solution; wherein the first and second binders bind to non-interfering epitopes on the analyte to form immune complexes; and wherein the immune complexes are captured on a first solid surface in contact with the solution via hybridization between a first nucleic acid tag ("first tag") conjugated to the first binder and a first nucleic acid capture probe ("first probe") coupled to the first solid surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface via dissociation of the hybridization between the first tag and the first probe; (4) introducing a second solid surface coupled with a second nucleic acid probe ("second probe") and recapturing the immune complexes onto the second solid surface via hybridization between the first tag and the second probe; (5) washing the second solid surface to remove unbound molecules; and (6) Detecting the immune complex. The present invention is also an assay method comprising:

[0346] In some embodiments, the second probe is the same as the first probe.

[0347] In some embodiments, the assay methods provided herein further comprise at least one additional recapture cycle between steps (5) and (6), comprising: releasing the immune complex from the solid surface on which it is captured; recapturing the immune complex on an additional solid surface coupled to a probe, which may be either the first probe, the second probe, or another nucleic acid probe that hybridizes to the first tag; and washing the additional solid surface to remove unbound molecules.

[0348] The use of nucleic acid hybridization as a capture / release mechanism offers several advantages to the assay methods disclosed herein, at least because the conditions for nucleic acid hybridization are quite different from those for protein binding. First, binding of the target analyte by the first binder and the second binder can be carried out in solution, allowing for fast binding kinetics and large sample inputs. Second, capture of immune complexes to solid surfaces by nucleic acid hybridization is usually more efficient, specific, and predictable. The sequence of the capture probe can be designed to specifically target the intended immune complex, which is useful in multiplexed assay formats. Third, dissociation of hybridization as a release mechanism can be achieved by changing the salt concentration in the buffer, which helps maximize the efficiency of release while maintaining the integrity of the immune complex. By intentionally designing a relatively weak hybridization bond between the capture probe and tag, either by a shorter segment or an A,T-rich sequence, it is possible to identify appropriate release buffer conditions under which the designated hybridization bond dissociates, releasing the immune complex while simultaneously allowing other hybridizations between the immune complex and longer or stronger complementary sequences to remain stable. Fourth, hybridization-based capture / release can be restarted and repeated multiple times without loss of efficiency and selectivity. Therefore, additional rounds of capture / release can be performed in the assay until a desired level of low background is reached. The robust capture / release mechanism ensures unprecedented predictability and reliability of the assay method. Another unexpected benefit is that nucleic acid capture probes coupled to the first and second surfaces help further reduce nonspecific encapsulation of the first and second binders due to their negatively charged oligonucleotide tags.

[0349] 5.2.2 Immune Complex Formation The assay method disclosed herein comprises step (1): forming an immune complex by mixing a first binder, a second binder, and a sample in a solution, wherein the first and second binders bind to non-interfering epitopes on the analyte, and the immune complex is captured on a first solid surface in contact with the solution through the bond between a first presenting group conjugated to the first binder and a first accepting group coupled to the first surface. In some embodiments, the first presenting group is a nucleic acid tag, and the first accepting group is a nucleic acid capture probe, wherein the probe or a fragment thereof is complementary to the tag or a fragment thereof.

[0350] As disclosed herein, the binder used in the present assay methods can be any molecule or portion of a molecule that binds to a specific target analyte. Thus, the binder can include any protein, peptide, nucleic acid, carbohydrate, lipid, or small molecule. In some embodiments, the binder includes an antibody. In some embodiments, the binder includes an antibody fragment. In some embodiments, the binder includes an antibody mimic. In some embodiments, the binder includes a small molecule.

[0351] The binder used in the assay methods disclosed herein can be conjugated to a presenting group (e.g., a nucleic acid tag). The binder and the presenting group can be linked either directly by a bond or indirectly by a linking group. When a linking group is used, such a group can be selected to provide covalent attachment between the presenting group and the binder and to maintain the desired binding affinity of the binder to its target analyte. The linking group can vary depending on the binder. When present, the linking group is usually biologically inert. A variety of linking groups are known to those skilled in the art and can be used in the assay methods disclosed herein. In some embodiments, the linking group comprises a spacer group terminating at either end with a reactive functional group that can be covalently bonded to the presenting group or the binder.

[0352] Spacer groups can include aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms such as oxygen (ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, and cyclic or acyclic systems that may contain heteroatoms. Spacer groups can also include ligands that bind to metals such that the presence of a metal ion coordinates two or more ligands to form a complex. Specific spacer components include: 1,4-diaminohexane, xylylenediamine, terephthalic acid, 3,6-dioxaoctanedioic acid, ethylenediamine-N,N-diacetic acid, 1,1'-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid), and 4,4'-ethylenedipiperidine. Potentially reactive functional groups include nucleophilic functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vinyls, ketones, epoxides, isocyanates, maleimides), functional groups capable of cycloaddition reactions, disulfide bond formation, or metal bonding. Specific examples include primary and secondary amines, hydroxamic acids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenyl esters, trifluoroethyl esters, glycidyl ethers, vinyl sulfones, and maleimides.Specific linker groups that may be used herein include heterofunctional compounds such as azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate, N-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-succinimidyl [4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate, 3-(2-pyridyldithio)propionic acid. Other examples include N-hydroxysuccinimide ester (SPDP) and 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC).

[0353] The binder / presenting group conjugates utilized in the assay methods disclosed herein can be prepared using any method known in the art. In some embodiments, the presenting group (e.g., a nucleic acid tag) can be conjugated to the binder either directly or via a linking group. Components can be covalently linked to each other via functional groups, as known in the art, where such functional groups can be present on the components or can be introduced onto the components using one or more steps, such as oxidation, reduction, cleavage, etc. Functional groups that can be used to covalently link components include hydroxy, sulfhydryl, amino, etc. The specific moieties of the various components modified to provide covalent bonds can be selected so as not to substantially adversely interfere with the desired binding affinity of the component to the target analyte. If necessary and / or desired, specific moieties on the components can be protected using blocking groups, as known in the art. See, for example, Green & Wuts, Protective Groups in Organic Synthesis (John Wley & Sons) (1991); U.S. Patent No. 5,733,523.

[0354] When nucleic acid tag is used as display group, binder / display group conjugate can also be produced by using the in vitro protocol that produces nucleic acid-protein conjugate.The example of such in vitro protocol of interest includes: RepA-based protocol (see, for example, Fitzgerald, Drug Discovery Today (2000) 5:253-258 and WO 98 / 37186), ribosome display-based protocol (see, for example, Hanes et al., Proc. Natl Acad. Sci. USA (1997) 94:4937-42; Roberts, Curr Opin Chem Biol (1999) Jun;3:268-73; Schaffitzel et al., J Immunol Methods (1999) Dec 10;231:119-35; and WO 98 / 54312).

[0355] When used in the assay method disclosed herein, the acceptor group (e.g., nucleic acid capture probe) can be "coupled" to the solid surface by any means known in the art, either directly or indirectly, for example, through a linking group such as those described above for linking the presenting group and the binder.For example, the acceptor group can be coupled to the solid surface by covalent linkage (e.g., chemical crosslinking) or non-covalent association, for example, streptavidin-biotin-based coupling (biotin is provided in one domain and streptavidin is provided in the other domain).In some embodiments, for example, the solid surface is the surface of a magnetic bead, which can be coupled with a nucleic acid capture probe as a presenting group, for example, through the bond between a carboxylic acid on the magnetic bead and the nucleic acid capture probe.In some embodiments, the solid surface can be covalently coupled with protein A / G as a presenting group.

[0356] The presenting group and the accepting group can be any binding pair disclosed herein or otherwise known in the art, including, but not limited to, an antigen and an antibody to the antigen (including fragments, derivatives, or mimetics thereof), a ligand and its receptor, complementary strands of nucleic acid, biotin and avidin (or streptavidin or neutravidin), a lectin and a carbohydrate (or vice versa). Additional binding pairs of "presenting group" and "acceptor group" include fluorescein and anti-fluorescein, digoxigenin / anti-digoxigenin, and DNP (dinitrophenol) / anti-DNP (or vice versa). In some embodiments, the binding pair of "presenting group" and "acceptor group" are complementary strands of nucleic acid and are referred to as a "tag" and a "probe." In some embodiments, the binding pair of "presenting group" and "acceptor group" is an antigen and an antibody, or an antigen and an antibody fragment.

[0357] As described above, a sample that can be assayed in the assay methods disclosed herein can be a material or mixture of materials containing one or more components of interest. In some embodiments, the sample is derived from a biological source. For example, the sample can be obtained from a subject and can be a biological tissue or fluid obtained, arrived at, or collected in vivo or in situ. Exemplary samples include biological fluids, such as blood samples, urine samples, plasma samples, saliva samples, cerebrospinal fluid samples, semen samples, sputum samples, mucus samples, dialysate samples, intestinal fluid samples, synovial fluid samples, and serous samples. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a saliva sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a cerebrospinal fluid sample. Exemplary samples include tissue samples. The tissue sample can be a liquid tissue sample. The tissue sample can be a homogenized tissue sample. The tissue sample can be obtained from a diseased tissue. In some embodiments, the sample is a cancer sample.

[0358] The solid surface may include any support known in the art that can be used for immobilizing molecules. In some embodiments, the solid surface may be any surface suitable for attaching nucleic acids and facilitating the assay process. Examples of solid surfaces include beads (e.g., magnetic beads, xMAP® beads), particles, colloids, single surfaces, tubes, chips, multiwell plates, microtiter plates, slides, membranes, cuvettes, gels, and resins. Exemplary solid surfaces include the surface of magnetic particles and the wells of a microtiter plate. When the solid phase is a particulate material (e.g., beads), it can be distributed in the wells of a multiwell plate to enable parallel processing. In some embodiments, the solid surface is the surface of magnetic beads. The magnetic beads can be coupled with a presentation group. In some embodiments, the magnetic beads can be carboxylate-modified, amine-blocked, oligo(dT)-coated, streptavidin-coated, protein A / G-coated, or silica-coated. In some embodiments, the solid surface is a well of a microtiter plate. In some embodiments, the first solid surface and the second solid surface are the same. In some embodiments, the first solid surface and the second solid surface are different. In some embodiments, both the first solid surface and the second solid surface used in the assay methods disclosed herein are the surfaces of magnetic particles. In some embodiments, both the first solid surface and the second solid surface used in the assay methods disclosed herein are the surfaces of microtiter plates.

[0359] As described above, the analyte measured in the assay methods disclosed herein can be any biological molecule. In some embodiments, the analyte is a protein analyte. In some embodiments, the analyte is a peptide analyte. In some embodiments, the analyte is a complex comprising at least two molecules. In some embodiments, the analyte is a protein complex comprising at least two proteins. In some embodiments, the analyte is a binding pair of two proteins. In some embodiments, the analyte is a macromolecular complex comprising at least one protein and at least one nucleic acid. In some embodiments, the analyte is a nucleic acid analyte.

[0360] In some embodiments, the analyte is a binding pair of two different molecules, where a first binder binds to one molecule of the binding pair and a second binder binds to the other molecule of the binding pair.

[0361] In some embodiments, the analyte is a binding pair of two different proteins, wherein a first binder binds to one protein of the binding pair and a second binder binds to the other protein of the binding pair. Thus, provided herein is an assay method for detecting protein-protein interactions in a sample, comprising the steps of: (1) mixing a first binder, a second binder, and the sample in a solution, wherein the first binder binds to one protein of the binding pair and the second binder binds to the other protein of the binding pair to form an immune complex, and wherein the immune complex is captured on the first solid surface in contact with the solution via binding between a first presenting group conjugated to the first binder and a first accepting group coupled to a first surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (4) introducing a second solid surface and recapturing the immune complex via binding between a second presenting group conjugated to the second binder and a second accepting group coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. The assay method includes:

[0362] In some embodiments, the first presenting group is a first nucleic acid tag ("first tag") and the first accepting group is a first nucleic acid capture probe ("first probe"), where the first probe or a fragment thereof is complementary to the first tag or a fragment thereof. In some embodiments, the second presenting group is a second nucleic acid tag ("second tag") and the second accepting group is a second nucleic acid capture probe ("second probe"), where the second probe or a fragment thereof is complementary to the second tag or a fragment thereof.

[0363] In some embodiments, provided herein is an assay method for detecting protein-protein interactions in a sample, comprising the steps of: (1) mixing a first binder, a second binder, and the sample in a solution; wherein the first binder binds to one protein of the binding pair and the second binder binds to the other protein of the binding pair to form an immune complex; and wherein the immune complex is captured on the first solid surface in contact with the solution via binding between a first presenting group conjugated to the first binder and a first accepting group coupled to a first surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (4) introducing the immunocomplex to a second solid surface coupled to a second acceptor group, and recapturing the immunocomplex to the second solid surface via the bond between the first presenting group and the second acceptor group; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. The assay method includes:

[0364] In some embodiments, the first presenting group is a first nucleic acid tag ("first tag") and the first acceptor group is a first nucleic acid capture probe ("first probe"), where the probe or a fragment thereof is complementary to the tag or a fragment thereof. In some embodiments, the second acceptor group is a second nucleic acid capture probe ("second probe"), where the probe or a fragment thereof is complementary to the tag or a fragment thereof.

[0365] The analyte can be a nucleic acid molecule (e.g., DNA and RNA). In some embodiments, the analyte is a DNA molecule. In some embodiments, the analyte is an RNA molecule. The assay methods provided herein can detect nucleic acid molecules directly in samples such as plasma and urine, without the need for nucleic acid isolation. As shown in Figure 15, the nucleic acid analyte can be hybridized and captured on a first surface, released into solution, and recaptured on a second surface, while the target-probe complex remains intact throughout the assay procedure. Thus, in some embodiments, the assay methods provided herein can be used to detect nucleic acid analytes. For example, provided herein is an assay method for detecting an analyte in a nucleic acid sample, comprising the following steps: (1) mixing a first binder, a second binder, and the sample in solution, wherein the first and second binders bind to non-interfering epitopes on the nucleic acid analyte to form an immune complex, and wherein the immune complex is captured on a first solid surface in contact with the solution via binding between a first presenting group conjugated to the first binder and a first accepting group coupled to the first surface; wherein the first and second binders of the nucleic acid analyte are nucleic acids complementary to various fragments of the nucleic acid analyte; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (4) introducing a second solid surface and recapturing the immune complex via binding between a second presenting group conjugated to the second binder and a second accepting group coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. The assay method includes:

[0366] In some embodiments, provided herein is an assay method for detecting an analyte in a nucleic acid sample, comprising the steps of: (1) mixing a first nucleic acid tag ("first tag"), a second nucleic acid tag ("second tag"), and the sample in solution, where fragments of the first tag and fragments of the second tag are each complementary to different fragments of the nucleic acid analyte and form immune complexes; wherein the immune complex is captured on a first solid surface in contact with the solution via hybridization between the first tag and a first nucleic acid capture probe ("first probe") coupled to the first solid surface, wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface via dissociation of the hybridization between the first tag and the first probe; (4) introducing the immunocomplex to a second solid surface and recapturing the immunocomplex on the second solid surface via hybridization between the second tag and a second nucleic acid capture probe ("second probe") coupled to the second solid surface; (wherein the second probe or a fragment thereof is complementary to the second tag or a fragment thereof); (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. The assay method includes:

[0367] The method can be used for high-throughput analysis of multiple targets by next-generation sequencing (NGS), including analysis of mutations, methylations, translocations, fusions, and / or copy number variations, etc. Alternatively, selected targets can be analyzed by qPCR, digital PCR, or other nucleic acid analysis techniques.

[0368] Step 1 of the assay method provided herein can be carried out in a variety of different approaches. For example, in some embodiments, a first binder can be pre-bound to a first surface that captures the analyte when contacted with a sample. In some other embodiments, a second binder can be added after or simultaneously with the sample, and immune complexes can be formed on the surface through multiple addition / incubation / washing steps.

[0369] Thus, in some embodiments, step (1) comprises forming an immune complex in solution before capturing the immune complex on a first solid surface. In some embodiments, step (1) comprises pre-capturing a first binder on a first solid surface before capturing the immune complex on the first solid surface. In some embodiments, in step (1), the immune complex is formed in solution and simultaneously captured on the first solid surface.

[0370] In some embodiments, a first binder is pre-bound to a first surface, a sample is added to the solution, and the analyte is allowed to bind to the first binder. Then, a second binder is added to bind to the analyte, forming an immune complex composed of the analyte and the binder. In some embodiments, a first binder is pre-bound to a first surface, and the sample and second binder are added simultaneously to form the immune complex. Those skilled in the art will understand that immune complexes can be formed using various approaches disclosed herein, or variations thereof with various addition / incubation / washing sequences.

[0371] (5.2.3 Alternative Capture Probe Configurations) As noted above, provided herein is an assay method for detecting an analyte in a sample, comprising the steps of: (1) mixing a first binder, a second binder, and the sample in solution, wherein the first and second binders bind to non-interfering epitopes on the analyte and form immune complexes, and wherein the immune complexes are captured on a first solid surface in contact with the solution via hybridization between a first tag conjugated to the first binder and a first probe coupled to the first surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface via dissociation of the hybridization between the first tag and the first probe; (4) introducing the immune complex to a second solid surface and recapturing the immune complex via binding between a second tag conjugated to a second binder and a second probe coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. The assay method includes:

[0372] Provided herein is an assay method for detecting an analyte in a sample, comprising: (1) mixing a first binder, a second binder, and the sample in solution; wherein the first and second binders bind to non-interfering epitopes on the analyte to form immune complexes; and wherein the immune complexes are captured on a first solid surface in contact with the solution via hybridization between a first nucleic acid tag ("first tag") conjugated to the first binder and a first nucleic acid capture probe ("first probe") coupled to the first solid surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface via dissociation of the hybridization between the first tag and the first probe; (4) introducing a second solid surface coupled with a second nucleic acid probe ("second probe") and recapturing the immune complexes onto the second solid surface via hybridization between the first tag and the second probe; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. : It is also an assay method comprising:

[0373] In addition to the direct capture configurations shown in Figures 4A-4D and 6A, Figure 6B shows an example of indirect capture in which the capture probe contains two segments: one complementary to the binder's tag and another complementary to another capture probe linked to the surface. Advantages of the indirect capture approach include, for example, that the sequence on the capture probe directly coupled to the solid surface can be universal (e.g., polyT or A), thus providing flexibility in multiplexed assays. Figure 6C shows another exemplary approach to indirect capture in which the capture probe is biotinylated and the surface is coated with streptavidin or avidin. Therefore, the capture probe is coupled to the surface by highly specific biotin-streptavidin / avidin binding.

[0374] Therefore, in some embodiments, direct capture is used in the assay method provided herein, in which the first probe is directly coupled to the first solid surface (FIG. 6A). In some embodiments, the second probe is directly coupled to the second solid surface. In some embodiments, the first probe is directly coupled to the first solid surface, and the second probe is directly coupled to the second solid surface.

[0375] In some embodiments, indirect capture is used in the assay method provided herein, where the first probe hybridizes with a universal probe that is directly coupled to a first solid surface, and the fragment of the first probe is complementary to the universal probe or a fragment thereof (Figure 6B).In some embodiments, the second probe hybridizes with a universal probe that is directly coupled to a second solid surface, and the fragment of the second probe is complementary to the universal probe or a fragment thereof.In some embodiments, the first probe hybridizes with a universal probe that is directly coupled to a first solid surface, and the second probe hybridizes with a universal probe that is directly coupled to a second solid surface.

[0376] In some embodiments, indirect capture is used in the assay methods provided herein, where the first probe is conjugated with biotin that binds to streptavidin or avidin that is directly coupled to the first solid surface (FIG. 6C). In some embodiments, the second probe is conjugated with biotin that binds to streptavidin or avidin that is directly coupled to the second solid surface. In some embodiments, the first probe is conjugated with biotin that binds to streptavidin or avidin that is directly coupled to the first solid surface, and the second probe is conjugated with biotin that binds to streptavidin or avidin that is directly coupled to the second solid surface.

[0377] In some embodiments, cooperative capture can be used in the assay methods described herein. As shown in Figure 6D, the capture probes simultaneously hybridize to shorter segments of the nucleic acid tags of binders 1 and 2. The assay conditions can be set so that the complementary segments are relatively short, so that the tags of the individual binders cannot stably hybridize to the capture probes alone. When both binders bind to the target protein and form an immune complex, the tags of the two binders cooperatively hybridize to the capture probes with sufficient strength to be stably captured on the surface. This approach also reduces non-specific binders that are not part of the immune complex, which reduces or even eliminates the need for release / recapture rounds.

[0378] Thus, in some embodiments, direct cooperative capture is used in the assay methods provided herein, in which a first tag and a second tag are cooperatively captured on a solid surface in step (1) (FIG. 6D). Provided herein is an assay method for detecting an analyte in a sample, comprising the following steps: (1) mixing a first binder, a second binder, and the sample in solution, wherein the first and second binders bind to non-interfering epitopes on the analyte and form immune complexes, and wherein the immune complexes are captured on a solid surface in contact with the solution via hybridization between a first nucleic acid tag ("first tag") conjugated to the first binder and a nucleic acid capture probe ("probe") coupled to the surface, and between a second nucleic acid tag ("second tag") conjugated to the second binder and the probe; (2) washing the solid surface to remove unbound molecules; and (3) detecting the immune complex. The assay method includes:

[0379] The cooperative capture can be repeated to further reduce non-specific binding. In some embodiments, the immune complexes are released from the first solid surface by dissociating the cooperative hybridization between the first and second tags and the probe, and are cooperatively recaptured onto a second solid surface, where the second solid surface is also coupled to the probe.

[0380] Cooperative capture can take various forms. In some embodiments, the continuous fragment of the probe consists of a first fragment and an adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first tag and the second tag are linked to form a linked nucleic acid, the junction region of the linked nucleic acid is complementary to the continuous fragment of the first probe (FIG. 7A). In some embodiments, the first tag and the second tag are also cooperatively captured on a second solid surface. In some embodiments, the continuous fragment of the second probe consists of a first fragment and an adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first tag and the second tag are linked to form a linked nucleic acid, the junction region of the linked nucleic acid is complementary to the continuous fragment of the second probe. In some embodiments, the second probe is the same as the first probe. In some embodiments, the immune complex is captured on a second solid surface through various mechanisms. For example, the second surface can be coupled with an antibody that binds to the first binder or the second binder.

[0381] In some embodiments, a first fragment of the first probe is complementary to a first tag or a fragment thereof, and another second fragment of the first probe is complementary to a second tag or a fragment thereof (FIGS. 6D and 7B). In some embodiments, the first tag and the second tag are also cooperatively recaptured to a second solid surface. In some embodiments, a first fragment of the second probe is complementary to a first tag or a fragment thereof, and another second fragment of the second probe is complementary to a second tag or a fragment thereof. In some embodiments, the second probe is the same as the first probe. In some embodiments, the complementary region comprises the unconjugated or "free" ends of the first and second tags, i.e., the ends that are not conjugated to the respective binders (FIG. 6D). In some embodiments, the complementary region does not comprise the unconjugated ends of the first and second tags (FIG. 7B). In some embodiments, the complementary region comprises the conjugated ends of the first and second tags (FIG. 7B). In some embodiments, the second probe is the same as the first probe. In some embodiments, the immune complex is captured on a second solid surface through various mechanisms. For example, the second surface can be coupled with an antibody that binds to the first binder or the second binder.

[0382] In some embodiments, direct cooperative capture is used in the assay methods provided herein, where a first solid surface is coupled to both a first probe and a second probe (Figure 7C). Provided herein is an assay method for detecting an analyte in a sample, comprising the following steps: (1) mixing a first binder, a second binder, and the sample in solution, wherein the first and second binders bind to non-interfering epitopes on the analyte and form immune complexes, and wherein the immune complexes are captured on a solid surface in contact with the solution via hybridization between a first nucleic acid tag ("first tag") conjugated to the first binder and a first nucleic acid capture probe ("first probe") coupled to the surface, and hybridization between a second nucleic acid tag ("second tag") conjugated to the second binder and a second nucleic acid capture probe ("second probe") coupled to the surface; (2) washing the solid surface to remove unbound molecules; and (3) detecting the immune complex. The assay method includes:

[0383] In some embodiments, the first tag and the second tag are also cooperatively recaptured to a second solid surface, where the second solid surface is also coupled to both the first probe and the second probe. In some embodiments, the first solid surface is coupled to the first probe and an additional probe, and the second solid surface is coupled to the second probe and an additional probe, where both the first probe and the second probe can hybridize to the first tag and the additional probe and to the second tag. In some embodiments, the second probe is the same as the first probe. In some embodiments, the immune complex is captured to the second solid surface through various mechanisms. For example, the second surface can be coupled to an antibody that binds to the first binder or the second binder.

[0384] The complementary sequence between the nucleic acid tag and the nucleic acid capture probe is designed to promote specific hybridization between the tag and the probe and subsequent dissociation of the hybridized immune complex.In one embodiment, a sequence with few or no G / Cs can be used, which has an appropriate length, can provide sufficient strength in hybridization, and can dissociate under low salt conditions without destroying the immune complex when released.In some embodiments, a sequence containing only poly T / A or poly "TA" / "AT" can be used.As those skilled in the art will understand, any sequence with suitable hybridization strength can be used.

[0385] In some embodiments, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of the complementary fragments between the nucleic acid tags and nucleic acid capture probes used in the assay methods provided herein are adenine ("A") and thymine ("T") pairs. In some embodiments, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of the complementary fragments between the first tag and the first probe are A and T pairs. In some embodiments, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of the complementary fragments between the second tag and the second probe are A and T pairs. In some embodiments, more than 80% of the complementary fragments between the first tag and the first probe are A and T pairs, and more than 80% of the complementary fragments between the second tag and the second probe are A and T pairs. In some embodiments, the complementary fragment of the first tag and the first probe comprises an A and T pair, and the complementary fragment of the second tag and the second probe comprises an A and T pair.

[0386] In some embodiments, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of the complementary fragments between the universal probe and the nucleic acid capture probe used in the assay methods provided herein are adenine ("A") and thymine ("T") pairs. In some embodiments, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of the complementary fragments between the universal probe and the first probe are A and T pairs. In some embodiments, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of the complementary fragments between the universal probe and the second capture probe are A and T pairs. In some embodiments, more than 80% of the complementary fragments between the universal probe and the first probe are A and T pairs, and more than 80% of the complementary fragments between the universal probe and the second probe are A and T pairs. In some embodiments, the universal probe and the first probe complementary fragment comprise an A and T pair, and the universal probe and the second probe complementary fragment comprise an A and T pair.

[0387] In some embodiments, the complementary fragment of the nucleic acid tag and the nucleic acid capture probe used in the assay methods provided herein consists of 10 to 30 base pairs, 10 to 25 base pairs, 12 to 20 base pairs, or 10 to 16 base pairs. In some embodiments, the complementary fragment of the nucleic acid tag and the nucleic acid capture probe used in the assay methods provided herein consists of 10 to 25 base pairs. In some embodiments, the complementary fragment of the nucleic acid tag and the nucleic acid capture probe used in the assay methods provided herein consists of 12 to 20 base pairs. In some embodiments, the complementary fragment of the nucleic acid tag and the nucleic acid capture probe used in the assay methods provided herein consists of 12 to 16 base pairs. In some embodiments, the complementary fragment of the nucleic acid tag and the nucleic acid capture probe used in the assay methods provided herein consists of 12 to 14 base pairs. In some embodiments, the complementary fragment of the first tag and the first probe consists of 10 to 25 base pairs. In some embodiments, the complementary fragment of the second tag and the second probe consists of 10 to 25 base pairs. In some embodiments, the complementary fragment between the first tag and the first probe consists of 12 to 20 base pairs. In some embodiments, the complementary fragment between the second tag and the second probe consists of 12 to 20 base pairs. In some embodiments, the complementary fragment between the first tag and the first probe consists of 12 to 16 base pairs. In some embodiments, the complementary fragment between the second tag and the second probe consists of 12 to 16 base pairs.

[0388] (5.2.4 Detection) After sufficient removal of non-specific binders that are not part of the immune complex by the capture / release mechanism, the immune complex can be detected. Thus, the assay methods provided herein include detecting the immune complex in step (6).

[0389] In some embodiments, the immune complexes are detected simultaneously as they are captured on the solid surface. The solid surface can be a second surface. If at least one additional capture / release cycle is included between steps (5) and (6), the solid surface can also be another solid surface. In some embodiments, the immune complexes are released from the solid surface into solution and detected in the solution.

[0390] In some embodiments, the assay methods provided herein comprise detecting a first binder of the immune complex. In some embodiments, the assay methods provided herein comprise detecting a second binder of the immune complex. In some embodiments, the assay methods provided herein comprise detecting a first presenting group conjugated to the first binder of the immune complex. In some embodiments, the assay methods provided herein comprise detecting a second presenting group conjugated to either the first binder or the second binder of the immune complex. In some embodiments, the immune complex is detected via a detectable marker conjugated to the first binder. In some embodiments, the immune complex is detected via a detectable marker conjugated to the second binder.

[0391] In some embodiments, the detectable marker is a nucleic acid that can be amplified and detected by polymerase chain reaction (PCR) (FIG. 2). By converting protein analyte detection into a nucleic acid reporter, immuno-PCR technology can be used to harness the power of nucleic acid technology for protein detection (e.g., US Pat. No. 5,665,539). As shown in FIGS. 8B and 8D, a nucleic acid segment pre-conjugated to a detection binder can be used as a reporter of the immune complex, and PCR is used to amplify the reporter and generate a detectable signal. In some embodiments, the nucleic acid tag used for capture / release can be detected, and no additional detectable marker is required. In some embodiments, a first tag is used for detection by PCR. In some embodiments, a second tag is used for detection by PCR.

[0392] Immune complexes can be detected by any method known in the art. For example, detection of immune complexes can be achieved using an antibody that specifically binds to the first binder, the second binder, the first presentation group, or the second presentation group. The detection antibody can be labeled with an enzyme, including, for example, horseradish peroxidase, alkaline phosphatase, or β-galactosidase, that can convert a colorless or nearly colorless substrate or co-substrate into a highly colored product or a product that can form a colored complex with a chromogen. Alternatively, the detection system can utilize an enzyme that emits light in the presence of an appropriate substrate. The amount of product formed can be detected visually, spectrophotometrically, electrochemically, fluorescently, or luminometrically and compared with a similarly treated control. The detection system can also utilize a radioactively labeled antibody, in which case the amount of immune complex is quantified by scintillation counting or gamma counting. Other detection systems that may be used include those based on the use of protein A from Staphylococcus aureus Cowan strain I, protein G from a group C Staphylococcus species (strain 26RP66).

[0393] In some embodiments, the immune complexes are detected by immunofluorescence. In some embodiments, the immune complexes are detected by detecting a detectable marker conjugated to the binder. The detectable marker can be a fluorescent labeling agent. The labeling agent can be a secondary antibody. The detectable marker can be a colorimetric detection reagent, a fluorescent detection reagent, or a chemiluminescent detection reagent. Colorimetric detectable markers can include PNPP (p-nitrophenyl phosphate), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), or OPD (o-phenylenediamine). Fluorescent detectable markers can include QuantaBlu™ or QuantaRed™ (Thermo Scientific, Waltham, MA). Luminescent detectable markers can include luminol or luciferin. In some embodiments, detectable markers can include a trigger (e.g., H2O2) and a tracer (e.g., isoluminol-conjugate).

[0394] The secondary antibody can be, for example, an anti-human IgA, anti-human IgD, anti-human IgE, anti-human IgG, or anti-human IgM antibody. The secondary antibody can be a monoclonal or polyclonal antibody. The secondary antibody can be derived from any mammalian organism, including mouse, rat, hamster, goat, camel, chicken, rabbit, and others. The secondary antibody can also be recombinant. The secondary antibody can be conjugated to an enzyme (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), luciferase, etc.) or a dye (e.g., a colorimetric dye, a fluorescent dye, a fluorescence resonance energy transfer (FRET) dye, a time-resolved (TR)-FRET dye, etc.). In some embodiments, the secondary antibody can be conjugated to a fluorescein (FITC)-based dye, such as fluorescein isothiocyanate. In some embodiments, the secondary antibody can be conjugated to Alexa Fluor® 488 (Life Technologies).

[0395] Methods and protocols for performing immunoassays and biophysical protein-interaction assays are well known in the art (see, e.g., Wild D., The Immunoassay Handbook, Elsevier Science, 4th Edition (2013); Fu H., Protein-Protein Interactions, Humana Press, 4th Edition (2004)).

[0396] 5.2.4.1 Generation of Nucleic Acid Reporters As shown in Figures 8A and 8C, in some embodiments, nucleic acid tags can be conjugated to binders used in the assay methods disclosed herein. Nucleic acid tags can be used to generate nucleic acid reporters that enable highly sensitive detection. As described above, provided herein is a two-capture binder assay method for detecting an analyte in a sample, comprising: (1) mixing a first binder, a second binder, and the sample in solution, wherein the first and second binders bind to non-interfering epitopes on the analyte and form immune complexes, and wherein the immune complexes are captured on a first solid surface in contact with the solution via hybridization between a first tag conjugated to the first binder and a first probe coupled to the first surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface via dissociation of the hybridization between the first tag and the first probe; (4) introducing the immunocomplex to a second solid surface and recapturing the immunocomplex via binding between a second tag conjugated to the second binder and a second probe coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. The assay method includes:

[0397] Provided herein is a capture binder assay method for detecting an analyte in a sample, comprising: (1) mixing a first binder, a second binder, and the sample in solution; wherein the first and second binders bind to non-interfering epitopes on the analyte to form immune complexes; and wherein the immune complexes are captured on a first solid surface in contact with the solution via hybridization between a first nucleic acid tag ("first tag") conjugated to the first binder and a first nucleic acid capture probe ("first probe") coupled to the first solid surface; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface via dissociation of the hybridization between the first tag and the first probe; (4) introducing a second solid surface coupled with a second nucleic acid probe ("second probe") and recapturing the immune complexes onto the second solid surface via hybridization between the first tag and the second probe; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. An assay method comprising:

[0398] In some embodiments of the one-capture binder assay method, the second binder is also conjugated to a second nucleic acid tag (a "second tag").

[0399] In both the two-capture binder assay method and the one-capture binder assay method, after sufficient removal of nonspecific first and second binders that are not part of the immune complex by the capture / release mechanism, a nucleic acid reporter can be generated using the first tag and the second tag. The nucleic acid reporter can take a variety of forms. For example, as shown in Figure 4B(e), the first tag and the second tag can be ligated to generate the nucleic acid reporter. As will be understood by those skilled in the art, any of the reporter generation methods disclosed herein or otherwise known in the art can be employed in this step, including, for example, ligation, polymerization extension, or cooperative hybridization.

[0400] In some embodiments, the second capture need not be releasable, and the nucleic acid reporter can be generated by the immune complex captured on the second surface. Alternatively, the immune complex can be first released and returned to solution, after which the nucleic acid reporter is generated.

[0401] Proximity ligation assays (PLA) and proximity extension assays (PEA) are known in the art (e.g., US 6,511,809, US 6,878,515, US 7,306,904, US 9,777,315, US 10,174,366, WO 9700446, Greenwood C, Biomol. Det. & Quan. 4 (2015) 10-16). Proximity-based detection differs from immuno-PCR in that it relies on the simultaneous recognition of a target analyte by two nucleic acid conjugate binders to trigger the formation of an amplifiable product. Therefore, individual nucleic acid conjugate binders that are not part of an immune complex do not generate a report, thus avoiding background from single nonspecifically bound binders. In some embodiments, proximity ligation is used to generate nucleic acid reporters (FIG. 3A), in which the first tag and the second tag are sufficiently close to be ligated upon the formation of an immune complex, and the fragment of the ligation product consisting of the fragment of the first tag and the fragment of the second tag is used as an amplicon to generate a signal for detection. In some embodiments, proximity extension is used to generate nucleic acid reporters (FIG. 3B), in which the first tag and the second tag are sufficiently close to interact with each other upon the formation of an immune complex to form a duplex, thereby extending the 3' end of at least one nucleic acid tag of the duplex to generate an extension product that can be used as an amplicon to generate a signal for detection. In some embodiments, cooperative hybridization is used to generate nucleic acid reporters, in which the first tag and the second tag are sufficiently close to interact with each other upon the formation of an immune complex to form a hybridization product that can be used as an amplicon to generate a signal for detection.

[0402] Nucleic acid reporters can be generated using nucleic acid tags in step (1) of the assay methods provided herein. As shown in Figure 4B(e), reporters can be generated by directly linking a first tag and a second tag via proximity ligation, proximity extension, or cooperative hybridization. Alternatively, as shown in Figures 5A-5C, nucleic acid reporters can be generated using nucleic acid surrogates that can hybridize to nucleic acid tags. For example, in some embodiments, surrogate nucleic acids are used, where the first surrogate or a fragment thereof is complementary to the first tag or a fragment thereof, and the second surrogate or a fragment thereof is complementary to the second tag or a fragment thereof. In some embodiments, reporters can be generated by linking a first surrogate to a second tag via proximity ligation, proximity extension, or cooperative hybridization (Figure 5A). In some embodiments, reporters can be generated by linking a first tag to a second surrogate via proximity ligation, proximity extension, or cooperative hybridization (Figure 5B). Alternatively, the reporter can be generated by linking a first surrogate to a second surrogate via proximity ligation, proximity extension, or cooperative hybridization (Figure 5C). In some embodiments, the first surrogate and / or the second surrogate can be added after the immune complex is formed. In another embodiment, the first surrogate and the second surrogate are pre-hybridized with the first tag and the second tag, respectively, prior to immune complex formation.

[0403] Thus, in some embodiments, step (6) of the assay methods provided herein comprises linking a first tag and a second tag by proximity ligation, proximity extension, or cooperative hybridization to generate a nucleic acid reporter, and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second tag. In some embodiments, surrogate nucleic acids are used, wherein the first tag or a fragment thereof is complementary to the first surrogate or a fragment thereof, and the second tag or a fragment thereof is complementary to the second surrogate or a fragment thereof. In some embodiments, step (6) of the assay methods provided herein comprises linking a first tag and a second surrogate via proximity ligation, proximity extension, or cooperative hybridization to generate a nucleic acid reporter, and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second surrogate. In some embodiments, step (6) of the assay methods provided herein comprises linking a first surrogate and a second tag via proximity ligation, proximity extension, or cooperative hybridization to generate a nucleic acid reporter, and detecting a nucleic acid reporter comprised of a fragment of the first surrogate and a fragment of the second tag. In some embodiments, step (6) of the assay methods provided herein comprises linking a first surrogate and a second surrogate via proximity ligation, proximity extension, or cooperative hybridization to generate a nucleic acid reporter, and detecting a nucleic acid reporter comprised of a fragment of the first surrogate and a fragment of the second surrogate.

[0404] The assay methods illustrated in Figures 5A-5C and 8A-8B use proximity ligation, however, as noted above, proximity extension, cooperative hybridization, or other methods known in the art can also be used to generate nucleic acid reporters for detection.

[0405] 5.2.4.2 Detection of Nucleic Acid Reporters The reporter generated in the final step can be detected using any existing nucleic acid detection technology, including, but not limited to, PCR, quantitative PCR (qPCR), digital PCR (dPCR), or next-generation sequencing (NGS). In some embodiments, the detection is qualitative detection. In some embodiments, the detection is quantitative detection. In some embodiments, the nucleic acid reporter is detected by qPCR. In some embodiments, the nucleic acid reporter is detected by dPCR. In some embodiments, the nucleic acid reporter is detected by NGS.

[0406] In addition to these common techniques, other nucleic acid amplification / detection methods can be used, including, but not limited to, rolling cycle amplification (RCA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). Additional techniques capable of highly sensitive nucleic acid detection without target amplification can also be employed for detecting nucleic acid reporters in the assay methods disclosed herein. These include, but are not limited to, the QuantiGene assay from ThermoFisher Scientific (ThermoFisher (2019)), the SIMOA assay from Quanterix (Rissin DM (2010)), and the SMCxPRO™ from MilliporeSigma (MilliporeSigma (2019)). Thus, in some embodiments, the nucleic acid reporter is detected by rolling cycle amplification (RCA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), or the QuantiGene assay.

[0407] (5.2.5 Multiplexing) Because the reporters generated in the assay methods disclosed herein are nucleic acid molecules, they can incorporate unique sequences as identification barcodes (IDs) that can be decoded by DNA sequencing or other methods. A segment of the ID containing N base nucleotides can be up to 4 N A unique identification code can be generated. One approach is to incorporate an ID directly into the tag of one binder (FIG. 9A) or both binders (FIG. 9B), as shown in FIGS. 9A-9B. In some embodiments, the nucleic acid reporter contains an ID in the first tag. In some embodiments, the nucleic acid reporter contains an ID in the second tag. In some embodiments, the nucleic acid reporter contains a first ID in the first tag and a second ID in the second tag.

[0408] Figure 10 illustrates an indirect ID barcoding approach in which the ID is on a separate single-stranded nucleic acid molecule (i.e., a nucleic acid surrogate) hybridized to a corresponding tag on the binder. In some embodiments, a surrogate nucleic acid is used, where a first tag or fragment thereof is complementary to the first surrogate or fragment thereof, and a second tag or fragment thereof is complementary to the second surrogate or fragment thereof. In Figure 10A, only the first binder is indirectly identified by the first surrogate. A nucleic acid reporter is generated by ligating the first surrogate to a second tag. In Figure 10B, both the first binder and the second binder are indirectly identified. While ligation is exemplified in Figures 9A-9B and 10A-10B, reporters can also be generated by proximity extension, cooperative hybridization, and other methods known in the art, as disclosed above. Furthermore, annealing of the ID-bearing nucleic acid surrogate to its associated binder tag can occur during the reagent manufacturing process prior to the assay. Alternatively, this can be done as part of the assay.

[0409] In some embodiments, provided herein are assay methods in which the nucleic acid reporter contains an identifier in the first tag, the first surrogate, the second tag, or the second surrogate. In some embodiments, the nucleic acid reporter is composed of a fragment of the first tag and a fragment of the second surrogate, and contains an identifier in the first tag or the second surrogate. In some embodiments, the nucleic acid reporter contains a first identifier in the first tag and a second identifier in the second surrogate. In some embodiments, the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second tag, and contains an identifier in the first surrogate or the second tag. In some embodiments, the nucleic acid reporter contains a first identifier in the first surrogate and a second identifier in the second surrogate. In some embodiments, the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second surrogate, and contains an identifier in the first surrogate or the second surrogate. In some embodiments, the nucleic acid reporter contains a first identifier in the first surrogate and a second identifier in the second surrogate.

[0410] One application of the above-mentioned ID method is to detect and measure multiple different analytes in the same sample in parallel. In some embodiments, the nucleic acid reporters disclosed herein contain analyte-specific IDs ("target IDs"). Each analyte is assigned a unique ID. Thus, provided herein is an assay method comprising simultaneously detecting at least two analytes in a sample by simultaneously detecting the unique target IDs associated with each analyte. In some embodiments, the assay method provided herein simultaneously detects at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 analytes in a sample by simultaneously detecting the unique target IDs associated with each analyte.

[0411] In some embodiments, the analyte is a protein. In some embodiments, the analyte comprises at least one protein and at least one nucleic acid. The nucleic acid can be DNA or RNA. The fact that the assay methods provided herein can be used to analyze proteins, DNA, and RNA makes the methods an ideal platform for multi-omics analysis.

[0412] As shown in Figure 11, one ID is sufficient to reveal the identity of the target analyte or the immune complex formed with the analyte. Once the ID is incorporated into the reporter, it can be detected and quantified by existing multiplexed nucleic acid detection technologies, such as multiplexed qPCR, multiplexed digital PCR, or next-generation sequencing (NGS).

[0413] Incorporation of an ID into the nucleic acid reporter in the assay methods provided herein can also help improve assay specificity. As shown in Figure 12, each of the first and second binders is associated with a unique ID, and only signals generated from reporters containing the IDs of both binders are counted as true signals. This scheme can be used to reduce or eliminate false positive signals generated by cross-reactivity or nonspecific binding between various binding pairs. Thus, provided herein are assay methods for detecting an analyte in a sample by simultaneous detection of a first target ID and a second target ID associated with a first binder and a second binder, respectively.

[0414] The incorporation of IDs into nucleic acid reporters in the assay methods provided herein can also be used to detect interactions between molecules, such as protein-protein interactions. As shown in Figure 13, simultaneous detection and quantification of reporters containing IDs of two binders designed for various molecules will indicate the interaction and affinity of these two related molecules under assay conditions. In some embodiments, the assay methods provided herein detect protein-protein interactions. Thus, provided herein is an assay method for detecting an analyte in a sample, wherein the analyte is a binding pair of two different proteins; wherein a first binder binds to one protein and a second binder binds to the other protein of the binding pair; and wherein the binding pair is detected by simultaneous detection of the first target ID and the second target ID.

[0415] In addition to an analyte-specific "target ID," the nucleic acid reporters generated in the assay methods provided herein can also include a sample-specific "sample ID." As shown in Figures 14A-14C, when the assay methods provided herein are performed on a particular sample, a sample ID can be introduced at or before the reporter generation step during the assay to identify the sample. By incorporating such a sample ID into the reporter, reporters from many samples can be pooled together and read in parallel by NGS. In some embodiments, the sample ID can be carried on the nucleic acid independently of the binder tag and incorporated into the reporter during the ligation step shown in Figure 14A. Thus, in some embodiments, the nucleic acid reporter formed in each sample contains an ID that is the sample ID, where the sample ID is inserted between a first tag or surrogate and a second tag or surrogate.

[0416] In some embodiments, the nucleic acid reporter is composed of a fragment of a first tag and a fragment of a second tag, and contains a sample ID inserted between the first tag and the second tag. In some embodiments, the nucleic acid reporter is composed of a fragment of a first tag and a fragment of a second surrogate, and contains a sample ID inserted between the first tag and the second surrogate. In some embodiments, the nucleic acid reporter is composed of a fragment of a first surrogate and a fragment of a second tag, and contains a sample ID inserted between the first surrogate and the second tag. In some embodiments, the nucleic acid reporter is composed of a fragment of a first surrogate and a fragment of a second surrogate, and contains a sample ID inserted between the first surrogate and the second surrogate.

[0417] Alternatively, the sample ID can be incorporated onto a nucleic acid surrogate that is hybridized to the tag during the assay step shown in Figure 14B. Thus, in some embodiments, the nucleic acid reporter formed in each sample contains the sample ID in the first surrogate or the second surrogate. In some embodiments, the nucleic acid reporter is composed of a fragment of the first tag and a fragment of the second surrogate, and contains the sample ID in the second surrogate. In some embodiments, the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second tag, and contains the sample ID in the first surrogate. In some embodiments, the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second surrogate, and contains the sample ID in the first surrogate or the second surrogate.

[0418] Alternatively, the sample ID can be incorporated by ligation to a nucleic acid tag or its surrogate during the assay step shown in Figure 14C. Thus, in some embodiments, the nucleic acid reporter formed in each sample contains a sample ID ligated to a first tag or a second tag, or their respective surrogates. In some embodiments, the nucleic acid reporter is composed of a fragment of a first tag and a fragment of a second tag, and contains a sample ID ligated to the first tag or the second tag. In some embodiments, the nucleic acid reporter is composed of a fragment of a first tag and a fragment of a second surrogate, and contains a sample ID ligated to the first tag or the second surrogate. In some embodiments, the nucleic acid reporter is composed of a fragment of a first surrogate and a fragment of a second tag, and contains a sample ID ligated to the first surrogate or the second tag. In some embodiments, the nucleic acid reporter is composed of a fragment of a first surrogate and a fragment of a second surrogate, and contains a sample ID ligated to the first surrogate or the second tag. In some embodiments, the nucleic acid reporter is composed of a fragment of a first surrogate and a fragment of a second surrogate, and contains a sample ID ligated to the first surrogate or the second surrogate.

[0419] Thus, also provided herein are assay methods that include simultaneously detecting analytes in at least two samples by incorporating a unique sample ID into a nucleic acid reporter formed in each sample and simultaneously detecting the unique sample ID in the nucleic acid reporter associated with each sample. In some embodiments, the assay methods provided herein simultaneously detect analytes in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting the unique sample ID associated with each sample.

[0420] In some embodiments, the nucleic acid reporter generated in the assay methods provided herein can include both a target ID and a sample ID. In some embodiments, the nucleic acid reporter includes (a) a target ID in a first tag or first surrogate, or in a second tag or second surrogate, and (b) a sample ID that is (1) inserted between the first tag or surrogate and the second tag or surrogate, (2) contained in the first surrogate or second surrogate, or (3) ligated to the first tag or surrogate or the second tag or surrogate.

[0421] In some embodiments, the nucleic acid reporter is composed of a fragment of a first tag and a fragment of a second tag, and contains a target ID and a sample ID. The nucleic acid reporter can contain a target ID in the first tag or the second tag. The nucleic acid reporter can contain a first target ID in the first tag and a second target ID in the second tag. The nucleic acid reporter can contain a sample ID inserted between the first tag or the second tag.

[0422] In some embodiments, the nucleic acid reporter is composed of a fragment of the first tag and a fragment of the second surrogate, and contains a target ID and a sample ID. The nucleic acid reporter can contain a target ID in the first tag or the second surrogate. The nucleic acid reporter can contain a first target ID in the first tag and a second target ID in the second surrogate. The nucleic acid reporter can contain a sample ID inserted between the first tag and the second surrogate. The nucleic acid reporter can also contain a sample ID in the second surrogate.

[0423] In some embodiments, the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second tag, and contains a target ID and a sample ID. The nucleic acid reporter can contain a target ID in the first surrogate or the second tag. The nucleic acid reporter can contain a first target ID in the first surrogate and a second target ID in the second tag. The nucleic acid reporter can contain a sample ID inserted between the first surrogate and the second tag. The nucleic acid reporter can also contain a sample ID in the first surrogate.

[0424] In some embodiments, the nucleic acid reporter is composed of a fragment of a first surrogate and a fragment of a second surrogate, and contains a target ID and a sample ID. The nucleic acid reporter can contain a target ID in the first surrogate or the second surrogate. The nucleic acid reporter can contain a first target ID in the first surrogate and a second target ID in the second surrogate. The nucleic acid reporter can contain a sample ID inserted between the first surrogate and the second surrogate. The nucleic acid reporter can also contain a sample ID in the first surrogate or the second surrogate.

[0425] Accordingly, also provided herein is an assay method that includes simultaneously detecting at least two analytes in at least two samples by simultaneously detecting a unique sample ID associated with each analyte and a unique target ID and a unique sample ID associated with each sample.

[0426] In some embodiments, the assay methods provided herein simultaneously detect at least two analytes in at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter for each sample.

[0427] In some embodiments, the assay methods provided herein simultaneously detect at least three analytes in at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter for each sample.

[0428] In some embodiments, the assay methods provided herein simultaneously detect at least five analytes in at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter for each sample.

[0429] In some embodiments, the assay methods provided herein simultaneously detect at least 10 analytes in at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter for each sample.

[0430] In some embodiments, the assay methods provided herein simultaneously detect at least 20 analytes in at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter for each sample.

[0431] In some embodiments, the assay methods provided herein simultaneously detect at least 50 analytes in at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter for each sample.

[0432] In some embodiments, the assay methods provided herein simultaneously detect at least 80 analytes in at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter for each sample.

[0433] In some embodiments, the assay methods provided herein simultaneously detect at least 100 analytes in at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter for each sample.

[0434] As will be appreciated by those skilled in the art, various permutations and combinations of the nucleic acid-based capture and ID configurations described above and shown in Figures 4A-7C and 9A-14 can be used in the assay methods disclosed herein.

[0435] The nucleic acid reporters in the multiplexed assay methods disclosed herein can be detected by multiplexed qPCR, multiplexed digital PCR, or NGS. For example, in some embodiments, the nucleic acid reporters in the multiplexed assay methods disclosed herein can be detected by NGS. The use of NGS to detect nucleic acid reporters generated by the assay methods disclosed herein includes at least the following advantages: First, NGS can detect and read the sequences of nucleic acid molecules in a sample. By incorporating target and sample ID tags into the reporters as described herein, NGS can perform multiplexed detection on a very large scale. For example, NGS can read a pool of 100 samples containing 10 targets each (i.e., 1000-plex) in a single run. This significantly reduces the cost per data point. Second, NGS can count and sum the number of molecules with the same sequence to provide digital quantification at single-molecule resolution. Because the reporter sequences are pre-designed and short, NGS as a reporter detection method can be completed much faster and at a lower cost than de novo sequencing. Furthermore, a variety of error correction algorithms from communication theory, such as parity check, Hamming code (e.g., Bystrykh, PLoS ONE 7(5): e36852(2012)), and Levenshtein code (e.g., Buschmann, BMC Bioinformatics. 2013; 14: 272(2013)), can be used and applied herein to reduce false positive counts, so that NGS-based quantification can achieve high accuracy without repeated sequencing.

[0436] Because NGS is a single-molecule detection and counting method, sequencing devices impose an upper limit on the total number of molecules that can be sequenced. For example, the Illumina MiSeq system can perform 25 million reads per run, limiting the total number of molecules sequenced in a single run to 25 million. This limitation is not restrictive in most applications when targets are present at low concentrations or near the limit of detection (LOD). However, in multiplexed tests, some targets are known to be expressed at levels several orders of magnitude higher than others, consuming sequencing bandwidth without providing useful clinical / biological information. Therefore, it is necessary to intentionally reduce the signals generated from these abundant analytes while maintaining sensitivity to the remaining analytes in a multiplexed assay format.

[0437] The assay method provided herein further addresses this need and provides related advantages. In some embodiments, the assay method provided herein is capable of accurately and knownly reducing the number of reporter molecules generated from a high concentration of target analyte, thereby efficiently allocating a limited detection bandwidth to various target analytes. The assay method disclosed herein uses an acceptor group (e.g., a nucleic acid capture probe) to capture immune complexes onto a solid surface before reporters are generated, which provides a unique opportunity to selectively capture only a small portion of the immune complexes generated therefrom onto the surface, thereby reducing the signal of extremely large amounts of analytes.

[0438] For example, in some embodiments, binders can be conjugated in a known ratio with or without their presenting group (e.g., nucleic acid tag).For example, if a binder conjugated with a presenting group is mixed with the same binder without a presenting group at a concentration of 1%, only 1% of the immune complex can be captured on the surface, and the ratio of reporter to target analyte is 1%.Non-functional presenting groups, i.e., presenting groups that do not bind to acceptor groups, can also be used.For example, if only 0.1% of the first binders are conjugated to functional first presenting groups (e.g., first tags), and the remaining 99.9% of the first binders are conjugated to first presenting groups (e.g., first tags) that cannot be captured by first acceptor groups (e.g., first probes), the ratio of reporter to immune complex is 1:1000, which effectively reduces the signal generated by this analyte by 1000 times.

[0439] Another approach for such partial capture can be used: introducing a known portion of non-functional acceptor groups (e.g., nucleic acid capture probes). In the indirect capture method shown in Figures 6B and 6C, for example, a certain percentage of first capture probes can be included that do not have a segment complementary to the universal capture probe (Figure 6B) or are not biotinylated (Figure 6C). As a result, the same percentage of immune complexes cannot be captured on the surface and therefore cannot generate nucleic acid reporters for detection. For example, if the acceptor groups contain only 0.1% of functional molecules that can be coupled to the solid surface (the remaining 99.9% are non-functional mimic molecules), the reporter-to-immune complex ratio will also be 1:1000, effectively reducing the signal generated by this analyte by 1000-fold.

[0440] Therefore, the present specification also provides an assay method, which comprises adding a non-functional binder to the solution in step (1) to proportionally reduce the amount of analyte detected by the assay, wherein the non-functional binder competes with the first binder for binding to the analyte, but is either unconjugated or conjugated to a presenting group that does not bind to the first acceptor group. In some embodiments, the non-functional binder is unconjugated. In some embodiments, the non-functional binder is conjugated to a presenting group that does not bind to the first acceptor group. In some embodiments, the non-functional binder is conjugated to a nucleic acid tag that cannot hybridize with the first probe coupled to the first solid surface.

[0441] Thus, also provided herein is an assay method that includes adding a non-functional receiver to the solution in step (1) to proportionally reduce the amount of analyte detected by the assay, where the non-functional receiver competes with the first acceptor for binding to the first presentation group but is unable to be coupled to the first solid surface.

[0442] In some embodiments, the present invention also provides an assay method in which step (1) involves adding a non-functional binder to the solution to proportionally reduce the amount of analyte detected by the assay, wherein the non-functional binder competes with either the first binder or the second binder for binding to the analyte, but forms an immune complex that cannot be detected. In some embodiments, the assay method provided herein detects the immune complex by detecting a detectable marker conjugated to either the first binder or the second binder, and the non-functional binder is not conjugated to a detectable marker or is conjugated to a defective detectable marker that cannot generate a reporter for detection. In some embodiments, the immune complex is detected via a nucleic acid reporter, and the non-functional binder can be conjugated to a nucleic acid tag that lacks an appropriate segment for generating a nucleic acid reporter. Those skilled in the art will understand that various approaches, which are variations of the methods disclosed herein, can be used to proportionally reduce the signal generated by extremely large amounts of analyte in a sample, allowing for the simultaneous detection of multiple analytes that may exist at concentrations that differ by several orders of magnitude.

[0443] In addition to NGS, many other single-molecule detection technologies, such as digital PCR and SIMOA, have limitations in the total number of target molecules that can be read by the system in a single run (instrument bandwidth). The above method of precisely controlling the reporter / immunocomplex ratio may be applicable to all of these technologies to maximize the efficient use of system resources.

[0444] 5.2.6 Assay Methods Using Target ID and / or Sample ID In one aspect, provided herein is an assay method for detecting an analyte in a sample, comprising: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (3) generating a reporter from the immune complex based on the proximity between the first target label and the second target label; and (4) detecting the reporter, thereby detecting the analyte. An assay method comprising:

[0445] As described in Section 5.1, target labels can provide an identifier that can be associated with a particular label to facilitate detection and identification of the target molecule. Thus, in some embodiments of the methods provided herein, including those described in this section (Section 5.2.6) and the previous paragraph, each target label can include one or more target IDs. In one specific embodiment, a first target label includes a first target ID. In another embodiment, a second target label includes a second target ID. In yet another embodiment, a first target label includes a first target ID and a second target label includes a second target ID.

[0446] As further described in Section 5.2.4 above, in some embodiments of the methods provided herein, including those in this section (Section 5.2.6), a reporter can be generated based on the proximity between a first target label and a second target label. Thus, in one embodiment, the reporter comprises a first target ID. In another embodiment, the reporter comprises a second target ID. In yet another embodiment, the reporter comprises a first target ID and a second target ID. In a further embodiment, the reporter comprises a first target ID and a second target ID. In one embodiment, the reporter comprises a first target ID and a sample ID. In another embodiment, the reporter comprises a second target ID and a sample ID. In a further embodiment, the reporter comprises a first target ID, a second target ID, and a sample ID.

[0447] In one aspect, provided herein is an assay method for detecting an analyte in a sample, comprising: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (3) generating a reporter from the immune complex, wherein the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and (4) detecting the reporter, thereby detecting the analyte. An assay method comprising:

[0448] As will be apparent from the description of, inter alia, Section 5.2.4.1 and Figures 3A-3C, in various embodiments of the methods provided herein, a reporter can be generated based on a property of the immune complex that correlates with the specificity of binding between both binders and the analyte. In one embodiment of the methods provided herein, including that of Section 5.2.6, a reporter is generated based on the proximity between a first target label and a second target label. In another embodiment, a reporter is generated based on the proximity between a first target ID and a second target ID. In yet another embodiment, a reporter is generated based on the proximity between a first binder and a second binder.

[0449] In one aspect, provided herein is an assay method for detecting an analyte in a sample, comprising: (1) mixing a first binding moiety comprising a first binder, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; and (ii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) generating a reporter from the immune complex, wherein the reporter is generated based on proximity between the first target label and the second target label, and wherein the reporter includes (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and (3) detecting the reporter, thereby detecting the analyte. An assay method comprising:

[0450] As disclosed in Section 5.2.4 and Figure 8F, in some embodiments of the methods provided herein, including those in this section (Section 5.2.6), the reporter is a non-nucleic acid reporter. In other embodiments, the reporter is a nucleic acid reporter. Thus, in one specific embodiment, the methods provided herein include: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (3) generating a nucleic acid reporter from the immune complex based on the proximity between the first target label and the second target label; and (4) detecting the reporter, thereby detecting the analyte. Contains: In another specific embodiment, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (3) generating a nucleic acid reporter from the immune complex, wherein the nucleic acid reporter comprises (i) a first target ID, (ii) a second target ID, or (iii) both the first target ID and the second target ID; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0451] Additionally, as described above in Section 5.2.1.1, immune complexes can be released from a first surface and recaptured on a second surface to further increase the signal-to-noise ratio. Thus, in one embodiment, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (3) generating a reporter from the immune complex based on the proximity between the first target label and the second target label; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0452] In another embodiment, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group, and simultaneously generating a reporter from the immune complex based on the proximity between the first target label and the second target label; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0453] In yet another embodiment, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (3) generating a reporter from the immune complex based on the proximity between the first target label and the second target label; and (3a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0454] In yet another embodiment, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (3) generating a reporter from the immune complex, wherein the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0455] In yet another embodiment, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group, and simultaneously generating a reporter from the immune complex, wherein the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0456] In a further embodiment, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (3) generating a reporter from the immune complex, wherein the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; (3a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0457] Additionally, as described in Section 5.2.1.1 above, immune complexes can be released from a first surface and recaptured on a second surface to further increase the signal-to-noise ratio. Figures 21A-21F show exemplary schematic diagrams of such assay methods involving two captures. Thus, in one embodiment of the methods provided herein, the second binding moiety further comprises a second presenting group. In another embodiment, the method further comprises step 2(b) between steps 2(a) and (3):(2b) introducing a second solid surface and recapturing the immune complexes on the second solid surface via binding between the second presenting group and a second accepting group coupled to the second solid surface. Thus, in some embodiments, the methods provided herein include: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (3) generating a reporter from the immune complex based on the proximity between the first target label and the second target label; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0458] In other embodiments, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (3) generating a reporter from the immune complex, wherein the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0459] Additionally, the methods provided herein further comprise step 2(c) between step 2(b) and step (3):(2c) washing the second solid surface to remove unbound molecules. Thus, in some embodiments, the methods provided herein further comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (3) generating a reporter from the immune complex based on the proximity between the first target label and the second target label; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0460] In other embodiments, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (3) generating a reporter from the immune complex, wherein the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0461] Additionally, the methods provided herein further comprise step 2(d): releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group. Thus, in some embodiments, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (2d) releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group; (3) generating a reporter from the immune complex based on the proximity between the first target label and the second target label; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0462] In other embodiments, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (2d) releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group; (3) generating a reporter from the immune complex, wherein the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0463] In yet another embodiment, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (3) releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group, and generating a reporter from the immune complex based on the proximity between the first target label and the second target label; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0464] In yet another embodiment, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (3) releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group, and generating a reporter from the immune complex, wherein the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0465] Thus, in some embodiments, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a second target label; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (3) generating a reporter from the immune complex based on the proximity between the first target label and the second target label; (4) releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group; and (5) detecting the reporter, thereby detecting the analyte. Contains:

[0466] In other embodiments, the methods provided herein comprise: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (3) generating a reporter from the immune complex, wherein the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and (4) releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group; and (5) detecting the reporter, thereby detecting the analyte. Contains:

[0467] In one aspect, provided herein is an assay method for detecting an analyte in a sample, comprising: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to non-interfering epitopes on the analyte and form immune complexes; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (3) generating a nucleic acid reporter comprising the first target ID and the second target ID; and (4) detecting the nucleic acid reporter, thereby detecting the analyte. An assay method comprising:

[0468] As described above in Sections 5.2.1.1 and 5.2.1.2, immune complexes can be detected and nucleic acid reporters generated either on the solid surface or after release from the solid surface. Figures 20A-20D provide exemplary schematics of the generation of nucleic acid reporters (shown as the first surface in Figures 20B-20C), and Figure 20E provides corroborating data from such an exemplary capture and release assay, in which the analyte is detected by a signal determined by the target ID in the target label generated in the nucleic acid reporter. Thus, in one embodiment, the method provided herein further comprises step (2a) between steps (2) and (3): releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group. In one specific embodiment, the method provided herein comprises: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to non-interfering epitopes on the analyte; and form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (3) generating a nucleic acid reporter comprising the first target ID and the second target ID; and (4) detecting the nucleic acid reporter, thereby detecting the analyte. Contains:

[0469] Furthermore, as described in Section 5.2.1.1 above, immune complexes can be released from a first surface and recaptured on a second surface to further increase the signal-to-noise ratio. Figures 21A-21F show exemplary schematic diagrams of such an assay method involving two captures. Thus, in one embodiment of the methods provided herein, the second binding moiety further comprises a second presenting group. In another embodiment, the method further comprises steps 2(b) and 2(c) between steps 2(a) and (3): (2b) introducing a second solid surface and recapturing the immune complexes on the second solid surface via binding between the second presenting group and a second accepting group coupled to the second solid surface; and (2c) washing the second solid surface to remove unbound molecules. Thus, in some embodiments, the methods provided herein include: (1) mixing a first binding moiety comprising a first binder and a first presentation group, a second binding moiety comprising a second binder and a second presentation group, and a sample in a solution, wherein: (i) the first and second binders bind to non-interfering epitopes on the analyte; and form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (3) generating a nucleic acid reporter comprising the first target ID and the second target ID; and (4) detecting the nucleic acid reporter, thereby detecting the analyte. Contains:

[0470] Similarly, as described above in this section and in Sections 5.2.1.1 and 5.2.1.2, immune complexes can be detected and nucleic acid reporters generated either on the solid surface or after release from the solid surface. Figures 21G-21I provide exemplary schematics of capture and generation of nucleic acid reporters after release from first and second solid surfaces, and Figures 24A-24B and 25A-25C provide confirmatory data showing such exemplary capture and release assays, in which the analyte is detected by a signal determined by the target ID in the target label generated in the nucleic acid reporter. Thus, in one embodiment, the method provided herein further comprises step (2d): releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group. In one specific embodiment, the method provided herein comprises: (1) mixing a first binding moiety comprising a first binder and a first presentation group, a second binding moiety comprising a second binder and a second presentation group, and a sample in a solution, wherein: (i) the first and second binders bind to non-interfering epitopes on the analyte; and form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (2d) releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group; (3) generating a nucleic acid reporter comprising the first target ID and the second target ID; and (4) detecting the nucleic acid reporter, thereby detecting the analyte. Contains:

[0471] As further described in Section 5.2.5 above, in addition to an analyte-specific "target ID," the nucleic acid reporters generated in the assay methods provided herein can also include a sample-specific "sample ID." Figure 22 and Figures 23A-23B provide an exemplary schematic diagram for generating a nucleic acid reporter with a sample ID, and Figures 24A-24B and 25A-25C provide confirmatory data demonstrating such a multiplexed assay, in which an analyte is detected by a signal determined by the target ID and the sample ID in the target label generated in the nucleic acid reporter, thereby analyzing the sample by sample ID and analyzing the analyte by target ID. Thus, in one embodiment, the method provided herein further comprises step (2e): binding a sample label comprising an ID that is sample-specific ("sample ID") to (i) a first target label, (ii) a second target label, or (iii) both the first target label and the second target label. In one specific embodiment, the method provided herein comprises: (1) mixing a first binding moiety comprising a first binder and a first presentation group, a second binding moiety comprising a second binder and a second presentation group, and a sample in a solution, wherein: (i) the first and second binders bind to non-interfering epitopes on the analyte; and form an immune complex; (ii) the immune complex is captured on the first solid surface in contact with the solution via a bond between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules; (2a) releasing the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; (2b) introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between a second presenting group and a second accepting group coupled to the second solid surface; (2c) washing the second solid surface to remove unbound molecules; (2d) releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group; (2e) binding a sample label comprising a sample-specific ID ("sample ID") to (i) the first target label, (ii) the second target label, or (iii) both the first target label and the second target label. (3) generating a nucleic acid reporter comprising the first target ID and the second target ID; and (4) detecting the nucleic acid reporter, thereby detecting the analyte. Contains:

[0472] Each binding moiety can comprise one or more target labels. In one embodiment, the first binding moiety comprises one target label. In another embodiment, the first binding moiety comprises two target labels. In a further embodiment, the first binding moiety comprises three target labels. In yet another embodiment, the first binding moiety comprises four target labels. In yet another embodiment, the first binding moiety comprises five or more target labels. In one embodiment, the second binding moiety comprises one target label. In another embodiment, the second binding moiety comprises two target labels. In a further embodiment, the second binding moiety comprises three target labels. In yet another embodiment, the second binding moiety comprises four target labels. In yet another embodiment, the second binding moiety comprises five or more target labels. In some embodiments, the target labels between the first binding moiety and the second binding moiety are different. In certain embodiments, the target labels between the first binding moiety and the second binding moiety are the same. In some further embodiments, the target labels within the first binding moiety are different. In other embodiments, the target labels within the first binding moiety are the same. In some further embodiments, the target labels within the second binding moiety are different. In other embodiments, the target labels in the second binding moiety are the same.In some embodiments, the first binding moiety has any combination of target labels provided in this paragraph, and the second binding moiety has any combination of target labels provided in this paragraph.In one specific embodiment, the first binding moiety comprises a first target label, and the second binding moiety comprises a second target label, wherein the first target label and the second target label are different.In another specific embodiment, the first binding moiety comprises a first target label, and the second binding moiety comprises a second target label, wherein the first target label and the second target label are the same.

[0473] Alternatively, one of the two binding moieties may not have a target label. In one embodiment of the methods provided herein, including those in this Section 5.2.6, the first binding moiety lacks a target label. In another embodiment, the second binding moiety lacks a target label. In a further embodiment, the first binding moiety does not comprise a target label. In yet another embodiment, the second binding moiety does not comprise a target label. In one embodiment, the first binding moiety does not comprise a target label and the second binding moiety comprises one target label. In another embodiment, the first binding moiety comprises one target label and the second binding moiety does not comprise a target label. In yet another embodiment, the first binding moiety does not comprise a target label and the second binding moiety comprises two, three, four, five, or more target labels. In another embodiment, the first binding moiety comprises two, three, four, five, or more target labels and the second binding moiety does not comprise a target label. In one embodiment, the first binding moiety lacks a target label and the second binding moiety comprises one target label. In another embodiment, the first binding moiety comprises one target label and the second binding moiety lacks a target label. In yet another embodiment, the first binding moiety lacks a target label and the second binding moiety comprises two, three, four, five, or more target labels. In another embodiment, the first binding moiety comprises two, three, four, five, or more target labels and the second binding moiety lacks a target label.

[0474] Similarly, one of the two binding moieties may not include a presenting group. In one embodiment of the methods provided herein, including those in this Section 5.2.6, the first binding moiety lacks a presenting group. In another embodiment, the second binding moiety lacks a presenting group. In a further embodiment, the first binding moiety does not include a presenting group. In yet another embodiment, the second binding moiety does not include a presenting group. In one embodiment, the first binding moiety does not include a presenting group and the second binding moiety includes one presenting group. In another embodiment, the first binding moiety includes one presenting group and the second binding moiety does not include a presenting group. In one embodiment, the first binding moiety lacks a presenting group and the second binding moiety includes one presenting group. In another embodiment, the first binding moiety includes one presenting group and the second binding moiety lacks a presenting group.

[0475] Further, in some embodiments of the methods provided herein, including those in this section (Section 5.2.6) and the previous paragraph, each target label can comprise one or more target IDs. In one embodiment, a first target label comprises a first target ID. In another embodiment, a second target label comprises a second target ID. In some embodiments, the target IDs between a first target label and a second target label are different. In certain embodiments, the target IDs between a first target label and a second target label are the same. In one specific embodiment, a first target label comprises a first target ID and a second target label comprises a second target ID, wherein the first target ID and the second target ID are different. In another specific embodiment, a first target label comprises a first target ID and a second target label comprises a second target ID, wherein the first target ID and the second target ID are the same.

[0476] Similarly, as further described in Section 5.2.5 above, in some embodiments of the methods provided herein, including those in this section (Section 5.2.6), each nucleic acid reporter can include one or more target IDs and / or sample IDs. In one embodiment, the nucleic acid reporter formed in each sample contains a sample ID. In another embodiment, the nucleic acid reporter formed in each sample contains a target ID. In a further embodiment, the nucleic acid reporter formed in each sample contains a first target ID. In yet another embodiment, the nucleic acid reporter formed in each sample contains a second target ID. In yet another embodiment, the nucleic acid reporter formed in each sample contains a sample ID and a first target ID. In one embodiment, the nucleic acid reporter formed in each sample contains a sample ID and a second target ID. In another embodiment, the nucleic acid reporter formed in each sample contains a first target ID and a second target ID. In a further embodiment, the nucleic acid reporter formed in each sample contains a first target ID and a second target ID, wherein the nucleic acid reporter lacks a sample ID. In yet another embodiment, the nucleic acid reporter formed in each sample contains a first target ID, a second target ID, and a sample ID.

[0477] When the nucleic acid reporter is generated from an immune complex formed from a first binding moiety comprising a first target label, a second binding moiety comprising a second target label, an analyte, and / or a sample label, such immune complexes formed in the methods provided herein (including those in this section (Section 5.2.6)) can similarly comprise one or more target IDs and / or sample IDs. In one embodiment, the immune complexes formed in each sample contain a sample ID. In another embodiment, the immune complexes formed in each sample contain a target ID. In a further embodiment, the immune complexes formed in each sample contain a first target ID. In yet another embodiment, the immune complexes formed in each sample contain a second target ID. In yet another embodiment, the immune complexes formed in each sample contain a sample ID and a first target ID. In one embodiment, the immune complexes formed in each sample contain a sample ID and a second target ID. In another embodiment, the immune complexes formed in each sample contain a first target ID and a second target ID. In further embodiments, the immune complexes formed in each sample contain a first target ID and a second target ID, wherein the immune complexes lack a sample ID. In yet another embodiment, the immune complexes formed in each sample contain a first target ID, a second target ID, and a sample ID.

[0478] Furthermore, when a sample ID is included in the sample label and a target ID is included in the target label, the immune complexes formed in the methods provided herein (including those in this section (Section 5.2.6)) can comprise one or more target labels and / or sample labels. In one embodiment, the immune complexes formed in each sample contain a sample label. In another embodiment, the immune complexes formed in each sample contain a target label. In a further embodiment, the immune complexes formed in each sample contain a first target label. In yet another embodiment, the immune complexes formed in each sample contain a second target label. In yet another embodiment, the immune complexes formed in each sample contain a sample label and a first target label. In one embodiment, the immune complexes formed in each sample contain a sample label and a second target label. In another embodiment, the immune complexes formed in each sample contain a first target label and a second target label. In a further embodiment, the immune complexes formed in each sample contain a first target label and a second target label, wherein the immune complexes lack a sample label. In yet another embodiment, the immune complexes formed in each sample contain a first target label, a second target label, and a sample label.

[0479] As provided in Section 5.1, an ID includes both an original ID molecule and a derivative ID molecule containing information derived from, but not identical to, the original ID, so long as such derivative molecule or information can identify a particular target or sample or otherwise distinguish a particular target or sample from other targets or samples and associate with the intended target or sample. Specifically, when the ID is a nucleic acid barcode sequence, the nucleic acid ID can comprise the original nucleic acid barcode sequence and / or the reverse complement of the original nucleic acid barcode sequence, as both can distinguish and associate with the intended target or sample. Thus, in some embodiments, the first target ID in the reporter comprises or consists of the original sequence of the first target ID in the first binding moiety. In certain embodiments, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety. In one embodiment, the second target ID in the reporter comprises or consists of the original sequence of the second target ID in the second binding moiety. In other embodiments, the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety. In one embodiment, the sample ID in the reporter comprises or consists of the original sequence of the sample ID in the sample label. In another embodiment, the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label.

[0480] In one embodiment, the first target ID in the reporter comprises or consists of the original sequence of the first target ID in the first binding moiety, and the second target ID in the reporter comprises or consists of the original sequence of the second target ID in the second binding moiety. In one embodiment, the first target ID in the reporter comprises or consists of the original sequence of the first target ID in the first binding moiety, and the sample ID in the reporter comprises or consists of the original sequence of the sample ID in the sample label. In one embodiment, the second target ID in the reporter comprises or consists of the original sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the original sequence of the sample ID in the sample label.

[0481] In one embodiment, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety, and the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety. In one embodiment, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety, and the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label. In one embodiment, the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label.

[0482] In one embodiment, the first target ID in the reporter comprises or consists of the original sequence of the first target ID in the first binding moiety, and the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety. In one embodiment, the first target ID in the reporter comprises or consists of the original sequence of the first target ID in the first binding moiety, and the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label. In one embodiment, the second target ID in the reporter comprises or consists of the original sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label. In one embodiment, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety, and the second target ID in the reporter comprises or consists of the original sequence of the second target ID in the second binding moiety. In one embodiment, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety, and the sample ID in the reporter comprises or consists of the original sequence of the sample ID in the sample label. In one embodiment, the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the original sequence of the sample ID in the sample label.

[0483] In one embodiment, the first target ID in the reporter comprises or consists of the original sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the original sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the original sequence of the sample ID in the sample label.

[0484] In one embodiment, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the original sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the original sequence of the sample ID in the sample label. In one embodiment, the first target ID in the reporter comprises or consists of the original sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the original sequence of the sample ID in the sample label. In one embodiment, the first target ID in the reporter comprises or consists of the original sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the original sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label.

[0485] In one embodiment, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the original sequence of the sample ID in the sample label. In one embodiment, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the original sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label.

[0486] In one embodiment, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the original sequence of the sample ID in the sample label. In one embodiment, the first target ID in the reporter comprises or consists of the original sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label.

[0487] In one embodiment, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the original sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label. In one embodiment, the first target ID in the reporter comprises or consists of the original sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label.

[0488] In one embodiment, the first target ID in the reporter comprises or consists of the complementary sequence of the first target ID in the first binding moiety, the second target ID in the reporter comprises or consists of the complementary sequence of the second target ID in the second binding moiety, and the sample ID in the reporter comprises or consists of the complementary sequence of the sample ID in the sample label.

[0489] As is apparent from the present disclosure, in the methods provided herein, including those provided in this Section (Section 5.2.6), the presenting group in the first binding moiety and the presenting group in the second binding moiety can each be any embodiment of the presenting group provided in this disclosure, including those provided in Sections 3, 5.1, 5.2.1-5.2.6, 5.3.1-5.3.3, and 6. The combination of the presenting group in the first binding moiety and the presenting group in the second binding moiety can be any combination of any embodiment of the presenting group provided in this disclosure, including those provided in Sections 3, 5.1, 5.2.1-5.2.6, 5.3.1-5.3.3, and 6. In one embodiment of the methods provided herein, the first presentation group is a polypeptide fused to a first binder. In another embodiment of the method provided herein, the first presentation group is a polynucleotide conjugated to the first binder. In another embodiment of the method provided herein, the first presentation group is a chemical compound conjugated to the first binder. In one embodiment of the method provided herein, the second presentation group is a polypeptide fused to the second binder. In another embodiment of the method provided herein, the second presentation group is a polypeptide conjugated to the second binder. In another embodiment of the method provided herein, the second presentation group is a chemical compound conjugated to the second binder.

[0490] In some embodiments of the methods provided herein, the first presentation grou...

Claims

1. 1. An assay method for detecting an analyte in a sample, comprising: Step (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first accepting group coupled to the first solid surface; and (iii) the first binding moiety further comprises a first target label comprising a first identification barcode (“ID”) (“first target ID”) that is analyte-specific, and the second binding moiety further comprises a second target label comprising a second target ID; step (2) washing the first solid surface to remove unbound molecules; step (2a) liberating the immune complex from the first solid surface by breaking the bond between the first presenting group and the first accepting group; step (3) generating a reporter from the immune complex based on proximity between the first target label and the second target label, wherein the reporter is a nucleic acid and includes (i) the first target ID, (ii) the second target ID, or (iii) both the first target ID and the second target ID; and Step (4) detecting the reporter, thereby detecting the analyte: The assay method comprising:

2. 10. The assay method of claim 1, wherein the second binding moiety further comprises a second presentation group: Step (2b) between steps (2a) and (3): introducing a second solid surface and recapturing the immune complex onto the second solid surface via binding between the second presenting group and a second accepting group coupled to the second solid surface. and Step (2c) between step (2b) and step (3): washing the second solid surface to remove unbound molecules. The assay method further comprises:

3. The assay method of claim 2, further comprising step (2d): releasing the immune complex from the second solid surface by breaking the bond between the second presenting group and the second accepting group.

4. 4. The assay method of claim 3, further comprising step (2e): binding a sample label comprising a sample-specific ID ("sample ID") to (i) the first target label, (ii) the second target label, or (iii) both the first target label and the second target label.

5. The assay method of any one of claims 1 to 4, wherein step (4) further comprises PCR amplification of the nucleic acid reporter.

6. The assay method according to any one of claims 1 to 5, wherein step (1) comprises forming the immune complex in the solution before capturing the immune complex on the first solid surface.

7. (i) the first presenting group is a first nucleic acid tag ("first tag") and the first accepting group is a first nucleic acid capture probe ("first probe"); or (ii) the second presenting group is a second nucleic acid tag ("second tag") and the second accepting group is a second nucleic acid capture probe ("second probe"), the assay method of any one of claims 2 to 6.

8. (i) the first presenting group is a first nucleic acid tag ("first tag") and the first accepting group is a first nucleic acid capture probe ("first probe"); and (ii) the second presenting group is a second nucleic acid tag ("second tag") and the second accepting group is a second nucleic acid capture probe ("second probe"), the assay method of any one of claims 2 to 6.

9. (i) the first probe is conjugated to streptavidin or biotin that binds to avidin that is directly coupled to the first solid surface; or 9. The assay method of claim 7, wherein (ii) the second probe is conjugated to biotin that binds to streptavidin or avidin that is directly coupled to the second solid surface.

10. the analyte is a peptide or a protein, and (i) the first binder is an antibody or antibody fragment that specifically binds to the analyte; (ii) the second binder is an antibody or antibody fragment that specifically binds to the analyte; or (iii) Both (i) and (ii); The assay method according to any one of claims 1 to 9.

11. The assay method according to any one of claims 1 to 10, wherein the sample is a serum sample or a plasma sample.

12. (i) in step (3), the nucleic acid reporter is generated simultaneously with the capture of the immune complex onto the second solid surface; or (ii) In step (3), after the immune complex is released from the second solid surface, the nucleic acid reporter is generated: The assay method according to any one of claims 2 to 11.

13. 13. The assay method of any one of claims 1 to 12, wherein the first target label is indirectly bound to the first binder and the second target label is directly bound to the second binder.

14. 14. The assay method of any one of claims 1 to 13, wherein the first presenting group is directly bound to the first binder and the second presenting group is indirectly bound to the second binder.

15. 15. The assay method of any one of claims 1 to 14, wherein the first presentation group is non-covalently bound to the first target label and the second presentation group is non-covalently bound to the second target label.

16. The sample labeling comprises: (i) a double-stranded nucleic acid molecule containing two 5' overhangs; (ii) a double-stranded nucleic acid molecule containing two 3' overhangs; (iii) a double-stranded nucleic acid molecule comprising a 5' overhang and a 3' overhang; (iv) a double-stranded nucleic acid molecule comprising a 5' overhang and a blunt end; or (v) a double-stranded nucleic acid molecule containing a 3' overhang and a blunt end and the sample label is: (i) hybridizes to the first target label via the overhang of the sample label; (ii) hybridizing to the second target label via the overhang of the sample label; or (iii) Both (i) and (ii); The assay method according to any one of claims 4 to 15.

17. the sample label is a double-stranded nucleic acid molecule comprising a 5' overhang and a 3' overhang; the sample label hybridizes to the first target label via the overhang of the sample label; and the sample label hybridizes with the second target label via the protrusion of the sample label; The assay method according to any one of claims 4 to 15.

18. The assay method according to any one of claims 7 to 17, wherein step (3) comprises linking the first tag and the second tag to generate the nucleic acid reporter, and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second tag.

19. The linking comprises linking (a) the first tag, (b) the second tag, and (c) (i) a double-stranded nucleic acid molecule containing two 5' overhangs; (ii) a double-stranded nucleic acid molecule containing two 3' overhangs; (iii) a double-stranded nucleic acid molecule comprising a 5' overhang and a 3' overhang; (iv) a double-stranded nucleic acid molecule comprising a 5' overhang and a blunt end; or (v) a double-stranded nucleic acid molecule containing a 3' overhang and a blunt end 20. The assay method of claim 18, comprising ligating one strand of the sample label which is

20. 20. The assay method of claim 19, wherein said linking comprises linking (c) between (a) and (b).

21. 21. The assay method of any one of claims 18 to 20, wherein the nucleic acid reporter is formed by proximity ligation.

22. Step (3) is generating the nucleic acid reporter by ligating a surrogate nucleic acid of the first tag and the second tag (a "second surrogate"), and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second surrogate; or generating the nucleic acid reporter by linking a surrogate nucleic acid of the first tag (a "first surrogate") to the second tag, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second tag; Including, wherein the first tag or fragment thereof is complementary to the first surrogate or fragment thereof, and the second tag or fragment thereof is complementary to the second surrogate or fragment thereof; and 21. The assay method of any one of claims 18 to 20, wherein said nucleic acid reporter is formed by proximity extension.

23. 23. The assay method of any one of claims 18 to 22, wherein the nucleic acid reporter comprises: (a) the first target ID or a surrogate nucleic acid for the first target ID (a "first target ID surrogate"); (b) the second target ID or a surrogate nucleic acid for the second target ID (a "second target ID surrogate"); and (c) a sample ID.

24. 24. The assay method of any one of claims 1 to 23, comprising simultaneously detecting at least two analytes in said sample by simultaneously detecting a unique target ID associated with each analyte.

25. 25. The assay method of any one of claims 1 to 24, wherein in step (4), detecting the analyte comprises simultaneous detection of the first target ID and the second target ID.

26. 26. The assay method of any one of claims 1 to 25, wherein step (1) further comprises mixing a reference analyte, wherein the reference analyte is an analyte not present in the sample.

27. 27. The assay method of any one of claims 4 to 26, further comprising pooling nucleic acid reporters from at least two samples prior to or concurrently with said detecting in step (4), and simultaneously detecting said analyte in said at least two samples by simultaneously detecting unique sample IDs in said nucleic acid reporters associated with each of said at least two samples.

28. 28. The assay method of any one of claims 1 to 27, wherein the reporters are detected by multiplexed qPCR, multiplexed digital PCR, or NGS.

29. 29. The assay method of any one of claims 26 to 28, wherein said detecting further comprises normalizing said reporter generated from said analyte of said sample to a reporter generated from said reference analyte.

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