Nucleic acid linked immune-sandwich assay (nulisa)

Abstract

To provide highly sensitive immunoassays that achieve detection with attomolar-level sensitivity.SOLUTION: Provided 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; (2) washing the first solid surface to remove unbound molecules; (3) releasing the immunocomplex from the first solid surface by disrupting the binding between the first presenting group and the first receiving group; (4) introducing a second solid surface and recapturing the immunocomplex via binding between the second presenting group conjugated to the second binder and the second receiving group coupled to the second solid surface; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application was filed on December 3, 2019, the disclosure of which is incorporated herein by reference in its entirety. This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 943,135, filed on 2004 / 01 / 14. (Sequence Listing) The present specification is submitted together with a computer readable form (CRF) copy of the sequence listing. The CRF titled 14582-003-228_SEQ_LISTING.txt, created on December 1, 2000, contains 13,423 bytes. No. 6,299,233, filed on Oct. 1, 2003, 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 methods for detecting target biomolecules or molecules. It relates to a highly sensitive immunoassay for the detection of complexes. [Background technology]

[0002] (2.Background) The detection of minute amounts of target molecules or molecular complexes in biological samples is important for both scientific research and clinical studies. With the invention of PCR and advances in polymerase chain reaction (PCR), detection of nucleic acids has become possible. Tremendous progress has been made in the past few decades in the analysis and analysis of The poor performance of current immunoassays for the detection of other biomolecules, such as proteins, remains a major concern. This is a technological bottleneck for many important applications, such as early cancer detection. is required for effective detection of analytes at 4,000 molecules / mL or ~7 attomolar concentrations ("aM"). This is beyond the capabilities of existing immunoassay techniques. There is a great need to improve assay performance. The present disclosure addresses these needs. and providing a highly sensitive immunoassay that addresses related issues and offers 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) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprising a second target label); (2) washing the first solid surface to remove unbound molecules; (3) detecting a receptor from the immune complex based on the proximity between the first target label and the second target label; generating a neutron; and (4) detecting the reporter, thereby detecting the analyte. : An assay method comprising:

[0004] Embodiment 2. (i) A first identification barcode ("ID") (" (ii) the second target label includes a second target identifier; or (iii) (i) and (ii). The assay method of embodiment 1, wherein the assay method is both.

[0005] Embodiment 3. (i) the reporter comprises a first target ID; or (ii) the reporter comprises a second target ID. or (iii) both (i) and (ii).

[0006] Embodiment 4. An assay method for detecting an analyte in a sample, comprising: (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the groups; and (iii) the first binding moiety binds to a first identification barcode (“ID”) that is analyte-specific (“Target ID”) ") and the second binding moiety comprises a second target ID. further comprising a second target label); (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 (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]

[0022] Embodiment 5. The reporter detects an immunoglobulin based on the proximity between a first target label and a second target label. The assay method of embodiment 4, wherein the complex is generated.

[0008]

[0022] Embodiment 6. The assay of any one of embodiments 1 to 5, wherein the reporter is a nucleic acid reporter. method.

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

[0010] Embodiment 8. Step (2a) between steps (2) and (3): Breaking the bond between the first presenting group and the first accepting group wherein the immunocomplexes are released from the first solid surface by , wherein step (2a) is before step (3), after step (3), or simultaneous with step (3), Any one of 7 assay methods.

[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 coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor groups. 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. Step (2d): By breaking the bond between the second presenting group and the second accepting group, 12. The method of claim 10 or 11, further comprising releasing the immune complexes from the second solid surface. Say method.

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

[0016]

[0023] Embodiment 14. (i) the first target ID and the second target ID are identical; or (ii) the first target ID and 14. The assay method of any one of embodiments 2 to 13, wherein said second target IDs are different.

[0017] Embodiment 15. Step (2e): A sample label comprising an ID ("sample ID") that is sample-specific is added to (i) a first label. (ii) to a second target label, or (iii) to both the first target label and the second target label. 2. The assay method of any one of the preceding embodiments, further comprising binding to said antibody.

[0018] Embodiment 16. The assay of embodiment 15, wherein the reporter formed in each sample comprises a sample ID. Method 1.

[0019] Embodiment 17. A polypeptide wherein the first presentation group is fused to a first binder, said first binder a polynucleotide conjugated to the first binder, or a polynucleotide conjugated to the first binder 10. The assay method of any one of the preceding embodiments, wherein the chemical compound is gated.

[0020] Embodiment 18. A polypeptide fused to a second binder, the second binder a polypeptide conjugated to the second binder, or a polypeptide conjugated to the second binder 18. The assay method of any one of embodiments 9 to 17, wherein the compound is a chemical compound.

[0021]

[0033] Embodiment 19. The foregoing embodiment, further comprising releasing the reporter from the immune complex. Any one of the assay methods.

[0022]

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

[0023]

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

[0024] Embodiment 22. Step (1) comprises capturing the immune complexes in solution before capturing the immune complexes on the first solid surface. 2. The assay method of any one of the preceding embodiments, comprising forming a complex.

[0025] Embodiment 23. Step (1) comprises administering a first binder to the first solid surface before the immune complex is formed on the first solid surface. 22. The method of any one of embodiments 1 to 21, comprising pre-capturing the antibody on said first solid surface. Say method.

[0026] Embodiment 24. In step (1), the immune complex is formed in solution and attached to a first solid surface. 22. The assay method of any one of embodiments 1 to 21, wherein simultaneous capture is performed.

[0027] Embodiment 25. (i) a first binder is directly bound to the analyte and a second binder is directly bound to the analyte; whether they are effectively combined; (ii) the first binder binds directly to the analyte and the second binder binds to the analyte; Indirectly bind or not; (iii) the first binder indirectly binds to the analyte and the second binder indirectly binds to the analyte; directly binds to; or (iv) the first binder indirectly binds to the analyte and the second binder indirectly binds to the analyte; Indirect binding: 10. The assay method of any one of the preceding embodiments.

[0028] Embodiment 26. (i) the first binder binds to a first primary antibody or fragment thereof that directly binds to the analyte; Will it fit? (ii) the second binder is a second primary antibody or fragment thereof that binds directly to the analyte; Combine; 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) releasing the immune complex from the solid surface on which it is captured; and recapturing the compound on a further solid surface coupled to an acceptor group of the compound of formula (I) above. At least one of steps (2) and (3) comprising: washing the surface to remove unbound molecules. additional recapture cycles; (ii) releasing the immune complex from the solid surface on which it is captured; recapture onto a further solid surface coupled to the first or second acceptor group; and washing the further solid surface to remove unbound molecules. at least one additional recapture cycle between (iii) Both (i) and (ii) 3. The assay method of any one of the preceding embodiments, further comprising:

[0031] Embodiment 29. Any of the liberating is by increasing the temperature to 70°C. The assay method of any one of embodiments 8 to 28,

[0032] Embodiment 30. (i) The first presentation group is a thioester group, a disulfide bond, or a cleavable (ii) the second presenting group is linked to the first accepting group via a thioester group, linked to a second acceptor group via a disulfide bond or a cleavable bond; or The assay method of any one of embodiments 1 to 29, wherein the method is both i) and (ii).

[0033] Embodiment 31. (i) The first presentation group is a photocleavable bond, a chemically cleavable bond, or an enzyme. (ii) the second presenting group is attached to the first accepting group via a photocleavable bond; to a second acceptor group via a bond that can be cleaved, chemically cleaved, or enzymatically cleaved. The assay method of embodiment 30, wherein the antibody binds to the target protein; or (i) and (ii) are both (i) and (ii).

[0034] Embodiment 32. (i) The first presentation group is coupled to the first receptor via a protein-protein interaction. (ii) the second presentation group binds to a second presentation group via a protein-protein interaction; or (i) and (ii) are both (i) and (ii). Method 1.

[0035] Embodiment 33. (i) The first presentation group is a biotin-mediated linkage to streptavidin or avidin. (ii) the second presentation group is a streptavidin or avidin-based antibody; or (i) and (ii), The assay method of any one of embodiments 1 to 29.

[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 is a 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 a 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 presentation group is a second a nucleic acid tag ("second tag") and a second acceptor group attached to a second nucleic acid capture probe ("second probe"); Robe) 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) the first probe is a protein and nucleic acid complex that specifically binds to the first tag; Ruka; (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 is 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; Ruka; (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 is 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 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] Embodiment 39. (i) the first probe is directly coupled to the first solid surface; or (ii) any of embodiments 34 to 38, wherein the second probe is directly coupled to the second solid surface. Any one assay method.

[0042] Embodiment 40. (i) the first probe is directly coupled to the first solid surface; and (ii) 39. Any of embodiments 34 to 38, wherein the second probe is directly coupled to the second solid surface. One assay method.

[0043] Embodiment 41. (i) A universal probe in which the first probe is directly coupled to the first solid surface. (ii) a second probe hybridizes directly to a second solid surface; 39. Any one of embodiments 34 to 38, wherein the hybridization with the coupled universal probe Two assay methods.

[0044] Embodiment 42. (i) A universal probe in which the first probe is directly coupled to the first solid surface. and (ii) a second probe is directly coupled to a second solid surface. 39. The method of any one of embodiments 34 to 38, wherein the hybridization of ... Assay methods.

[0045] Embodiment 43. (i) A stress reaction in which the first probe is directly coupled to the first solid surface. or (ii) conjugated to biotin, which binds to putavidin or avidin; The second probe may be streptavidin or ATP directly coupled to a second solid surface. 39. Any one of embodiments 34 to 38, wherein the antibody is conjugated to biotin, which binds to avidin. Two assay methods.

[0046] Embodiment 44. (i) A stress reaction in which the first probe is directly coupled to the first solid surface. (ii) a second protease inhibitor conjugated to avidin or biotin that binds to avidin; and The probe is coupled directly to a second solid surface using streptavidin or avidin. 39. The assay of any one of embodiments 34 to 38, wherein the assay is conjugated to biotin, which binds to ribonucleotides. Say method.

[0047] Embodiment 45. The first tag and the second tag are cooperatively attached to the first solid surface in step (1). The assay method of any one of embodiments 34 to 44, wherein the antibody is captured by

[0048] Embodiment 46. The first fragment of the first probe is complementary to the first tag or a fragment thereof, a second fragment of the first probe that is complementary to the second tag or a fragment thereof, 46. ​​The assay of embodiment 45, wherein the target region comprises the unconjugated ends of said first and said second tags. method.

[0049] Embodiment 47. The contiguous fragment of the first probe comprises a first fragment and an immediately adjacent second fragment. wherein when the first tag and the second tag are linked to form a linked nucleic acid, the the first probe so that the junction region of the ligated nucleic acid is complementary to the contiguous fragment of the first probe; the fragment of said first tag or a fragment thereof is complementary to said first tag or a fragment thereof, and the second fragment of said second tag or a fragment thereof is complementary to said second tag or a fragment thereof. The assay method of embodiment 45, wherein the fragment is complementary to a fragment of

[0050] Embodiment 48. The first fragment of the first probe is complementary to the first tag or a fragment thereof; and another second fragment of the first probe is complementary to a second tag or a fragment thereof; and 46. ​​The method of claim 45, wherein the complementary region does not include the unconjugated ends of the first and second tags. Assay methods.

[0051] Embodiment 49. The first solid surface is coupled to both the first probe and the additional nucleic acid probe. and the additional probe or a fragment thereof is complementary to the first tag or a fragment thereof; The assay method of embodiment 45.

[0052] Embodiment 50. The first tag and the second tag are cooperatively attached to a second solid surface in step (2b). 50. The assay method of any one of embodiments 35 to 49, wherein the antibody is captured by

[0053] Embodiment 51. The first fragment of the second probe is complementary to the first tag or a fragment thereof, The second fragment of the second probe is complementary to the second tag or a fragment thereof, wherein the complementary 51. The assay method of embodiment 50, wherein the region comprises the unconjugated ends of the first and second tags. Law.

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

[0055] Embodiment 53. The first fragment of the second probe is complementary to the first tag or a fragment thereof; and another second fragment of the second probe is complementary to a second tag or a fragment thereof; and 51. The method of embodiment 50, wherein the complementary region does not include the unconjugated ends of the first and second tags. Assay methods.

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

[0057] Embodiment 55. (i) The complementary fragment of the first tag and the first probe consists of 10 to 30 base pairs; (i i) the complementary fragment of the second tag and the second probe consists of 10 to 30 base pairs; or (i) and (ii) The assay method of any one of embodiments 34 to 54, wherein both

[0058] Embodiment 56. (i) The complementary fragment of the first tag and the first probe consists of 20 to 30 base pairs; (i i) the complementary fragment of the second tag and the second probe consists of 20 to 30 base pairs; or (i) and (ii) The assay method of any one of embodiments 34 to 55, wherein the method is both.

[0059] Embodiment 57. (i) the first tag comprises an A- and / or T-rich sequence, and the first probe (ii) the second tag contains a complementary A- and / or T-rich sequence; and the second probe comprises a complementary A- and / or T-rich sequence; or (i) and (ii) The assay method of any one of embodiments 34 to 56, wherein ii) and ii) are both

[0060] Embodiment 58. The binding between the first binder and the analyte is due to complementary A- and / or T-rich sequences. and the bond between the second binder and the analyte, thereby preventing release. In the step (2a) and / or (2d) of incubating the immunoconjugate, A and / or T are added to the immunoconjugate so as to stably maintain the immunoconjugate. The assay method of embodiment 57, wherein the rich sequence has a short length.

[0061] Embodiment 59. The analyte is a binding pair of two molecules; and wherein the first binder is a member of the binding pair. The aforementioned embodiment in which a first binder binds to one molecule and a second binder binds to the other molecule of the binding pair. The assay method of any one of the embodiments.

[0062] Embodiment 60. The analyte is a nucleic acid, and the first and second binders of the nucleic acid analyte are 10. The assay method of any one of the preceding embodiments, comprising nucleic acids complementary to various fragments of the Law.

[0063] Embodiment 61. The method of claim 61, wherein the analyte is a peptide or protein and (i) the first binder binds to the peptide or protein. (ii) the second binder is an antibody or antibody fragment that specifically binds to the analyte; or (i) and (ii), The assay method of any one of embodiments 1 or 59.

[0064] Embodiment 62. The assay of any one of the preceding embodiments, wherein the sample is a serum sample or a plasma sample. Say method.

[0065] Embodiment 63. (i) In step (3), the immune complex is captured on a first solid surface while simultaneously capturing a nucleic acid reporter. is generated; or (ii) in step (3), after the immune complex is released from the first solid surface, The parameter 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 simultaneously is generated; or (ii) in step (3), after the immune complex is released from the second solid surface, The parameter is generated: An assay method according to any one of embodiments 10 to 62.

[0067] Embodiment 65. (i) the first target label is directly bound to the first binder; or (ii) the 65. The first target label is indirectly bound to said first binder: Two assay methods.

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

[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 One 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 directly attached to the second binder; Two presenting groups are 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 method of embodiments 15 to 74, wherein the sample label is a single-stranded nucleic acid molecule ("single-stranded sample label"). Any one assay method.

[0078]

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

[0079] Embodiment 77. The sample label 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 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; detecting a nucleic acid reporter composed of a fragment of the first tag and a fragment of the second tag; (b) by linking a surrogate nucleic acid of the first tag and the second tag (a "second surrogate"); generating the nucleic acid reporter, which is comprised of a fragment of the first tag and a fragment of the second surrogate; detecting a nucleic acid reporter; (c) by linking a surrogate nucleic acid of the first tag (the "first surrogate") to the second tag. generating the nucleic acid reporter, which is comprised of the first surrogate fragment and the second tag fragment; detecting a nucleic acid reporter; or (d) linking the first surrogate with the second surrogate to generate the nucleic acid reporter. and detecting a nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second surrogate. Putting out; Contains: wherein the first tag or a fragment thereof is complementary to the first surrogate or a 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; detecting a nucleic acid reporter composed of a fragment of the first tag and a fragment of the second target label; thing; (b) linking the first tag to a surrogate nucleic acid of the second target label (a "second surrogate"); The nucleic acid reporter is generated by the first tag fragment and the second surrogate fragment. detecting the nucleic acid reporter formed; (c) linking a surrogate nucleic acid of the first tag ("first surrogate") to the second target label; The nucleic acid reporter is generated by the first surrogate fragment and the second target label fragment. detecting a nucleic acid reporter comprising: (d) linking the first surrogate with the second surrogate to generate the nucleic acid reporter. and detecting a nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second surrogate. Putting out; Contains: wherein the first tag or a fragment thereof is complementary to the first surrogate or a 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.

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

[0084] Embodiment 82. Step (3) comprises: (a) Linking a first target label to a second target label to generate a nucleic acid reporter. and a nucleic acid reporter composed of a fragment of the first target label and a fragment of the second target label. To detect; (b) linking a surrogate nucleic acid of the first target label to the second target label (a "second surrogate"); to generate the nucleic acid reporter, and to separate the fragments of the first target label and the second surrogate. detecting a nucleic acid reporter composed of a fragment; (c) linking a surrogate nucleic acid of the first target label (the "first surrogate") to the second target label; to generate the nucleic acid reporter, and to separate the fragments of the first surrogate and the second target label. detecting a nucleic acid reporter comprising a fragment; or (d) linking the first surrogate with the second surrogate to generate the nucleic acid reporter. and detecting a nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second surrogate. Putting out; Contains: wherein the first target label or a fragment thereof is complementary to the first surrogate or a 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 substitute therefor; and (b) a second tag or a second substitute therefor. and (c) a substitute nucleic acid for the single-stranded sample label ("sample surrogate") or one strand of the double-stranded sample label; (ii) (a) the first tag or a first surrogate thereof and (b) a second target label or a second surrogate thereof. and (c) a substitute nucleic acid for the single-stranded sample label (a "sample surrogate") or one of the double-stranded sample labels. one chain; (iii) (a) a first target label or a first surrogate thereof; and (b) the second tag or a second surrogate thereof. and (c) a substitute nucleic acid for the single-stranded sample label (a "sample substitute") or the double-stranded sample label. one strand of; or (iv) (a) the first target label or a first surrogate thereof; and (b) the second target label or a first surrogate thereof. a second surrogate and (c) a surrogate nucleic acid ("sample surrogate") for the single-stranded sample label or the double-stranded sample label. One chain of signs 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] Embodiment 84. In each of (i) to (iv), linking (c) between (a) and (b) The assay method of embodiment 83, comprising:

[0087]

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

[0088]

[0044] Embodiment 86. Any of embodiments 79 to 84, wherein the nucleic acid reporter is formed by proximity extension. One assay method.

[0089]

[0062] Embodiment 87. A nucleic acid reporter comprising: (a) a first target ID or a surrogate nucleic acid of said first target ID ("first (b) a second target ID or a surrogate nucleic acid for the second target ID (“second target ID surrogate”); 87. The assay method of any one of embodiments 79 to 86, comprising (a) a sample ID, (b) a sample replacement, and (c) a sample ID.

[0090]

[0062] Embodiment 88. The method of claim 88, wherein the unique target ID associated with each analyte is detected simultaneously. 88. Any one of embodiments 2 to 87, comprising simultaneously detecting at least two analytes of Assay methods.

[0091] Embodiment 89. In step (1), the analysis is performed by adding a non-functional binder to the solution. and proportionally reducing a signal from at least one of the non-functional The reactive binder competes with the first binder for binding to the analyte but binds to the first receptor group. The fluorophore is either not conjugated or conjugated to a non-conjugated presenting group. , The assay method of embodiment 88.

[0092]

[0062] Embodiment 90. In step (4), detecting the analyte comprises detecting a first target ID and a second target ID. 90. The assay method of any one of embodiments 2 to 89, comprising simultaneous detection of:

[0093] Embodiment 91. The aforementioned embodiment, further comprising mixing a reference analyte in step (1). Any one of the following assay methods.

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

[0096] Embodiment 94. The reference analyte is a viral protein, a bacterial protein, or an insect protein. The assay method of embodiment 93, wherein

[0097]

[0062] Embodiment 95. Simultaneously detecting a unique sample ID in a nucleic acid reporter associated with each sample. and simultaneously detecting the analyte in at least two samples. Any one of the four assay methods.

[0098] Embodiment 96. Prior to or simultaneously with the detection in step (4), nuclei from at least two samples are collected. The assay method of embodiment 95, further comprising pooling acid reporters.

[0099]

[0062] Embodiment 97. A unique sample ID in a nucleic acid reporter associated with each analyte in each sample. Simultaneous detection of unique target IDs allows for the identification of at least two molecules in at least two samples. 96. The assay method of any one of embodiments 15 to 95, comprising simultaneously detecting the precipitates.

[0100] Embodiment 98. Prior to or simultaneously with the detection in step (4), small amounts of nucleotides from at least two samples are collected. In some embodiments, the method further comprises pooling nucleic acid reporters for at least two analytes. Assay method for 97.

[0101] Embodiment 99. The nucleic acid reporter is capable of being used in multiplexed qPCR, multiplexed digital PCR, or the like. 10. The assay method of any one of the preceding embodiments, wherein the detection is by PCR or NGS.

[0102]

[0033] Embodiment 100. Any one of the preceding embodiments, wherein the nucleic acid reporter is detected by NGS. Assay method.

[0103]

[0033] Embodiment 101. The detecting comprises separating a reporter generated from an analyte of the sample from a reference analyte. 101. The method of any of embodiments 91 to 100, further comprising normalizing to a reporter generated from the One assay method.

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

[0105]

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

[0106] Embodiment 104. (1) a first binder and a first binding moiety including a first presenting group and a second binder and mixing the sample in solution with a second binding moiety comprising two presentation groups, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety contains a first identification barcode (“ID”) that is analyte-specific (“target”) and the second binding moiety further comprises a first target label comprising a second target ID. further comprising a second targeting label; (2) washing the first solid surface to remove unbound molecules; (3) disrupting the bond between the first presenting group and the first accepting group, thereby converting the immune complex into a first To release from a solid surface; (4) introducing a second solid surface and coupling a second presenting group to the second solid surface; recapturing the immune complex to the second solid surface via binding between the second acceptor group; (5) washing the second solid surface to remove unbound molecules; (6) A sample label containing a sample-specific ID ("sample ID") is added to (i) the first target label and (ii) the second target label. or (iii) to both the first target label and the second target label; (7) extracting nucleic acid fragments from the immune complex based on the proximity between the first target label and the second target label. generating a reporter, wherein the nucleic acid reporter is a reporter of the first target ID, the second target ID, including the target ID, and the sample ID); (8) pooling nucleic acid reporters from at least two samples; (9) disrupting the bond between the second presenting group and the second accepting group to thereby bind the immune complex. releasing from said second solid surface; (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. (i) A first identification barcode ("ID") in which the first target label is analyte-specific. (ii) the second target label includes a second target ID; or (iii) (i) and (ii) 106. The system of embodiment 105, wherein

[0109] Embodiment 107. (i) a first binder, a first presentation group, and a first identification barcode (" a first binding moiety comprising a first target label comprising a target ID); (ii) a second binding moiety comprising a second binder and a second target label comprising a second target ID; and Beauty (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]

[0044] Embodiment 108. The method of any one of embodiments 105 to 107, wherein the reporter is a nucleic acid reporter. The system described above.

[0111] Embodiment 109. The second binding moiety further comprises a second presenting group, wherein the system comprises a second accepting group. and wherein a second presenting group is attached to said second accepting group. The system described in any one of the preceding paragraphs.

[0112]

[0033] Embodiment 110. (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 method further comprises: The sample label is attached to (i) a first target label, (ii) a second target label, or (iii) the first target label. A system according to any one of embodiments 105 to 110, which binds both the target molecule and the second target label.

[0114]

[0062] Embodiment 112. (i) a first target ID and a second target ID, or (ii) the first target ID and the second target for proximity ligation or proximity extension to generate a nucleic acid reporter containing a sample ID and a sample ID. 112. The system of any one of embodiments 106 to 111, further comprising a reagent.

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

[0118] Embodiment 116. A first solid surface is coupled to a first acceptor group and a second solid surface is coupled to a second acceptor group. 116. The system according to embodiment 114 or 115, which is coupled to two acceptor groups.

[0119] Embodiment 117. A polypeptide wherein the first presentation group is fused to a first binder, said first A polynucleotide conjugated to a binder or a polynucleotide conjugated to said first binder 117. The system of any one of embodiments 105 to 116, which is a conjugated chemical compound.

[0120] Embodiment 118. The second presentation group is a polypeptide fused to a second binder, the second binder a polypeptide conjugated to said second binder, or The system according to any one of embodiments 109 to 117, wherein the system is a chemical compound.

[0121]

[0044] Embodiment 119. The method of embodiments 108 to 118, further comprising reagents for PCR amplification of the nucleic acid reporter. 10. The system of any one of claims 1 to 9.

[0122]

[0044] Embodiment 120. The method of embodiments 108 to 119, further comprising reagents for purifying the nucleic acid reporter. 10. The system of any one of claims 1 to 9.

[0123] Embodiment 121. (i) a first binder is directly bound to the analyte and a second binder is directly bound to the analyte; whether they are effectively combined; (ii) the first binder binds directly to the analyte and the second binder binds to the analyte; Indirectly bind or not; (iii) the first binder indirectly binds to the analyte and the second binder indirectly binds to the analyte; directly binds to; or (iv) the first binder indirectly binds to the analyte and the second binder indirectly binds to the analyte; Indirect binding: A system described in any one of embodiments 105 to 120.

[0124] Embodiment 122. (i) the first binder binds to a first primary antibody or fragment thereof that directly binds to the analyte; Will it fit? (ii) the second binder is a second primary antibody or fragment thereof that binds directly to the analyte; Combine; 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] Embodiment 124. (i) the first presentation group is a thioester group, a disulfide bond, or a cleavable (ii) the second presenting group is linked to the first accepting group via a thioester group, linked to a second acceptor group via a disulfide bond or a cleavable bond; or The system of any one of embodiments 105 to 123, wherein both i) and (ii) are true.

[0127] Embodiment 125. (i) The first presentation group is a photocleavable bond, a chemically cleavable bond, or an enzyme. (ii) the second presenting group is attached to the first accepting group via a photocleavable bond; to a second acceptor group via a bond that can be cleaved, chemically cleaved, or enzymatically cleaved. or both (i) and (ii).

[0128] Embodiment 126. (i) a first presentation group is coupled to a first receptor via a protein-protein interaction. (ii) the second presentation group binds to the second presentation group via a protein-protein interaction; or both (i) and (ii). The system described above.

[0129] Embodiment 127. (i) The first presentation group couples biotin to streptavidin or avidin. (ii) the second presentation group is bound to the first acceptor group via streptavidin or via biotin to avidin, to a second acceptor group; or both (i) and (ii). A system described in any one of embodiments 105 to 123.

[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 is a 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 a capture probe ("second probe"), A system described in any one of embodiments 105 to 123.

[0131] Embodiment 129. (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 presentation group is a second a nucleic acid tag ("second tag") and a second acceptor group attached to a second nucleic acid capture probe ("second probe"); 124. The system of any one of embodiments 109 to 123, wherein the nucleus is a nucleus-like protrusion ("nucleus-like protrusion").

[0132] Embodiment 130. (i) whether the first probe is a protein that specifically binds to the first tag; (ii) the first probe is a protein and nucleic acid complex that specifically binds to the first tag; Ruka; (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 is 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; Ruka; (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 is hybridizes to the second tag or a fragment thereof: 131. The system of embodiment 128 or 130.

[0134] Embodiment 132. The complementarity is at least 80%, at least 81%, at least 82%, at least 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 132. The system of embodiment 130 or 131, which is 98%, or at least 99% complementary.

[0135] Embodiment 133. (i) the first probe is directly coupled to the first solid surface; or or (ii) the second probe is directly coupled to the second solid surface, 33. The system of any one of claims 32.

[0136] Embodiment 134. (i) the first probe is directly coupled to the first solid surface; and (ii) 133.) The method of any one of embodiments 128 to 132, wherein the second probe is directly coupled to the second solid surface. 10. The system of any one of claims 1 to 9.

[0137] Embodiment 135. (i) A universal probe in which the first probe is directly coupled to the first solid surface. (ii) a second probe hybridizes directly to a second solid surface; 133. Any of embodiments 128 to 132, which hybridizes with a coupled universal probe. The system described in claim 1.

[0138] Embodiment 136. (i) A universal probe in which the first probe is directly coupled to the first solid surface. and (ii) a second probe is directly coupled to a second solid surface. 133. Any one of embodiments 128 to 132, wherein the hybridization is performed with a universal probe that is pulled. The system described.

[0139] Embodiment 137. (i) a stress reaction in which the first probe is directly coupled to the first solid surface. or (ii) conjugated to biotin, which binds to putavidin or avidin; The second probe may be streptavidin or ATP directly coupled to a second solid surface. 133. Any of embodiments 128 to 132, wherein the antibody is conjugated to biotin, which binds to avidin. The system described in claim 1.

[0140] Embodiment 138. (i) a stress reaction in which the first probe is directly coupled to the first solid surface. (ii) a second protease inhibitor conjugated to avidin or biotin that binds to avidin; and The probe is coupled directly to a second solid surface using streptavidin or avidin. 133. The method of any one of embodiments 128 to 132, wherein the antibody is conjugated to biotin, which binds to ribonucleotides. System of.

[0141] Embodiment 139. The embodiment in which the first tag and the second tag are cooperatively captured on a first solid surface. The system of any one of embodiments 128 to 138.

[0142]

[0062] Embodiment 140. The first fragment of the first probe is complementary to the first tag or a fragment thereof; a second fragment of the first probe that is complementary to a second tag or a fragment thereof, 140. The method of embodiment 139, wherein the complementary region comprises the unconjugated ends of the first and second tags. system.

[0143] Embodiment 141. The contiguous fragment of the first probe comprises a first fragment and an immediately adjacent second fragment. wherein when the first tag and the second tag are linked to form a linked nucleic acid, the the second probe so that the junction region of the linked nucleic acid is complementary to the contiguous fragment of the first probe; one 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. 140. The system of embodiment 139, which is complementary to a fragment thereof.

[0144]

[0062] Embodiment 142. The first fragment of the first probe is complementary to the first tag or a fragment thereof; and a second fragment of the first probe is complementary to a second tag or a fragment thereof; and

[0023] Embodiment 13, wherein said complementary regions do not include the unconjugated ends of said first and said second tags. 9 described system.

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

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

[0147]

[0062] Embodiment 145. The first fragment of the second probe is complementary to the first tag or a fragment thereof; a second fragment of the second probe is complementary to the second tag or a fragment thereof, 145. The system of embodiment 144, wherein the target region comprises the unconjugated ends of said first and said second tags. .

[0148] Embodiment 146. The contiguous fragment of the second probe comprises a first fragment and an immediately adjacent second fragment. wherein when the first tag and the second tag are linked to form a linked nucleic acid, the the first probe so that the junction region of the linked nucleic acid is complementary to the contiguous fragment of the second probe. one 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. 145. The system of embodiment 144, which is complementary to a fragment thereof.

[0149]

[0062] Embodiment 147. The first fragment of the second probe is complementary to the first tag or a fragment thereof; and another second fragment of the second probe is complementary to a second tag or a fragment thereof; and

[0023] Embodiment 14. wherein said complementary regions do not include the unconjugated ends of said first and said second tags. 4. The system described.

[0150] Embodiment 148. The second solid surface is coupled to both the second probe and the additional nucleic acid probe. and the additional probe or a fragment thereof is complementary to the second tag or a fragment thereof. 145. The system of embodiment 144.

[0151] Embodiment 149. (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 (i) and (ii) 149. The system of any one of embodiments 128 to 148, wherein

[0152] Embodiment 150. (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 (i) and (ii) 150. The system of any one of embodiments 128 to 149, wherein

[0153] Embodiment 151. (i) the first tag comprises an A- and / or T-rich sequence, and the first probe (ii) the second tag contains a complementary A- and / or T-rich sequence; the second probe comprises a complementary A- and / or T-rich sequence; or (i) The system of any one of embodiments 128 to 150, wherein (i) and (ii) are both:

[0154] Embodiment 152. The binding between the first binder and the analyte is due to complementary A- and / or T-rich sequences. and the bond between the second binder and the analyte. 152. The system of embodiment 151, wherein the rich sequence has a short length.

[0155] Embodiment 153. The analyte is a binding pair of two molecules; and wherein the first binder is a binding pair. and a second binder binds to the other molecule of the binding pair. 153. The system according to any one of claims 105 to 152.

[0156] Embodiment 154. The analyte is a nucleic acid, and the first and second binders of the nucleic acid analyte bind to the nucleic acid molecule. 154. The system according to any one of embodiments 105 to 153, comprising nucleic acids complementary to various fragments of the precipitate. .

[0157] Embodiment 155. The method of claim 15, wherein the analyte is a peptide or a protein and (i) the first binder binds to the peptide or protein. (ii) the second binder is an antibody or antibody fragment that specifically binds to the analyte; or (i) and (ii), The system of any one of embodiments 105 to 153.

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

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

[0160] Embodiment 158. (i) the second target label is directly bound to the second binder; or (ii) The second target label is indirectly bound to the second binder: The system described in any one of the preceding paragraphs.

[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. (i) the first presenting group is directly attached to the first binder; or (ii) the The first presenting group is indirectly attached to the first binder: any of embodiments 105 to 160. The system described in claim 1.

[0164] Embodiment 162. (i) the second presenting group is directly attached to the second binder; or (ii) the The second presenting group is indirectly attached to the second binder: any of embodiments 109 to 161. The system described in claim 1.

[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. Embodiments 106-16, wherein the sample label is a single-stranded nucleic acid molecule ("single-stranded sample label"). 7. The system of any one of claims 6.

[0170]

[0066] Embodiment 168. Embodiments 106-16, wherein the sample label is a double-stranded nucleic acid molecule ("double-stranded sample label"). 7. The system of any one of claims 6.

[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]

[0062] Embodiment 171. The system comprises: 170. The method of claim 106, wherein at least two analytes in a sample can be detected simultaneously. 10. The system of any one of claims 1 to 9.

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

[0177] Embodiment 175. The reference analyte is a viral protein, a bacterial protein, or an insect protein. 175. The system of embodiment 174, wherein the system is of a quality.

[0178]

[0062] Embodiment 176. The system simultaneously detects unique sample IDs in nucleic acid reporters associated with each sample. By using the method, it is possible to simultaneously detect analytes in at least two samples. The system of any one of embodiments 111 to 175.

[0179]

[0062] Embodiment 177. The system comprises: a unique nucleic acid reporter associated with each analyte in each sample; Simultaneous detection of sample ID and unique target ID allows for the identification of at least two samples. The method according to any one of embodiments 110 to 176, wherein two analytes can be detected simultaneously. system.

[0180] Embodiment 178. Multiplexed qPCR for detecting nucleic acid reporters. Any of embodiments 105 to 177, comprising reagents and / or equipment for automated digital PCR or NGS. 10. The system according to any one of claims 1 to 9.

[0181]

[0062] Embodiment 179. A method for detecting a nucleic acid reporter comprising the steps of: A system described in any one of embodiments 105 to 177.

[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 kit comprises a binding buffer, an immobilization buffer, a wash buffer, a release buffer. 181. The system of embodiment 180, comprising a buffer, or any combination thereof.

[0184] Embodiment 182. (1) A method for detecting a first binder, a first presentation group, and an analyte-specific first identification. (1) a first binding moiety comprising a first target label comprising a barcode (“ID”) (“target ID”); and (2) a second binding moiety comprising a first target label comprising a barcode (“ID”) (“target ID”); a second binder comprising the 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) a light-sensitive material; (5) a sample label containing a reagent for gating and a sample-specific ID ("sample ID"); and Contains reagents for quantitative PCR; where: (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 captures the first presenting group; arranged to be; (iii) the second accepting group is coupled to the second solid surface and captures the second presenting group; positioned to capture; (iv) the first target label is directly or indirectly bound to the first binder, and the second binder is is directly or indirectly bound to said second binder; (v) the first presenting group is directly or indirectly bound to the first binder, and the second a presenting group is attached directly or indirectly 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 kit comprises a binding buffer, an immobilization buffer, a wash buffer, a release buffer. 184. The system of embodiment 183, further comprising a buffer, or any combination thereof.

[0187] Embodiment 185. (1) A method for detecting a first binder, a first presentation group, and an analyte-specific first identification. (1) a first binding moiety comprising a first target label comprising a barcode (“ID”) (“target ID”); and (2) a second binding moiety comprising a first target label comprising a barcode (“ID”) (“target ID”); a second binder comprising the 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 sample label containing reagents for ligation and a sample-specific ID ("sample ID"). where: (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 captures the first presenting group; arranged to be; (iii) the second accepting group is coupled to the second solid surface and captures the second presenting group; positioned to capture; (iv) the first target label is directly or indirectly bound to the first binder, and the second is directly or indirectly bound to said second binder; (v) the first presenting group is directly or indirectly bound to the first binder, and the second a 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.

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

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

[0190] Provided herein is an assay method that uses two capture binders. In some embodiments, provided herein are methods for detecting an analyte in a sample. An assay method comprising: (1) Mixing a first binder, a second binder, and the sample in a solution (wherein the first and second binders bind to non-interfering epitopes on the analyte; and the immune complex and the immunocomplex is bound to a first presenting group conjugated to the first binder. a first surface coupled to the first receptor group in contact with the solution through a bond between the first receptor group and the first surface coupled to the first receptor group; trapped on solid surfaces); (2) washing the first solid surface to remove unbound molecules; (3) disrupting the bond between the first presenting group and the first accepting group, thereby converting the immune complex into a first To release from a solid surface; (4) introducing a second solid surface and a second presentation group conjugated to the second binder; and a second acceptor group coupled to the second solid surface. recapturing the body; (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 capturing the immune complexes on a first solid surface prior to capturing the immune complexes on a first solid surface. In some embodiments, the step comprises forming the immune complex in solution. (1) is a method for binding a first binder to a first solid surface before capturing an immune complex on the first solid surface. In some embodiments, in step (1), the immunoglobulin is pre-captured onto a body surface. Immunocomplexes are formed in solution and simultaneously captured on a first solid surface.

[0192] In some embodiments, the assay methods provided herein comprise detecting the immune complexes by: releasing the immune complex from the solid surface on which it is captured; recapturing the compound on a further solid surface coupled with an acceptor group, and and washing the surface to remove unbound molecules. Further included is an additional recapture cycle.

[0193] In some embodiments, (i) the first presenting group is a thioester group, a disulfide bond, or (ii) the second presenting group is attached to the first accepting group via a bond that is cleavable or that can be broken down; , a thioester group, a disulfide bond, or a cleavable bond to a second acceptor group or both (i) and (ii).

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

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

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

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

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

[0200] In some embodiments, the first tag and the second tag are are cooperatively trapped on the solid surface of

[0201] In some embodiments, the first fragment of the first probe is a first tag or a fragment thereof. a second fragment of the first probe that is complementary to a second tag or a fragment thereof; wherein the complementary regions include the unconjugated ends of the first and second tags. nothing.

[0202] In some embodiments, the contiguous fragment of the first probe comprises the first fragment and the immediately adjacent fragment. wherein the first tag and the second tag are linked to form a linked nucleic acid When the junction region of the linked nucleic acid is formed, the junction region of the linked nucleic acid is complementary to the continuous fragment of the first probe. The first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the first tag or a fragment thereof, such that The piece is complementary to the second tag or a fragment thereof.

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

[0204] In some embodiments, the first solid surface comprises a first probe and a second probe. It is coupled with both.

[0205] In some embodiments, (i) the complementary fragment of the first tag and the first probe is between 10 and 2 (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 methods for detecting an analyte in a sample. An assay method for detecting a marker comprising: (1) mixing a first binder, a second binder, and the sample in a solution; The first and second binders bind to non-interfering epitopes on the analyte to form immune complexes. and wherein the immunocomplex comprises a first binder conjugated to the first binder. a nucleic acid tag ("first tag") and a first nucleic acid capture protocol coupled to a first solid surface; The first probe contacts the solution through hybridization between the first probe and the second probe. trapped on a solid surface); (2) washing the first solid surface to remove unbound molecules; (3) detecting hybridization between the first tag and the first probe in the immune complex; liberating from said first solid surface via dissociation; (4) introducing a second solid surface coupled with a second nucleic acid probe ("second probe"); and the immune complex is subjected to hybridization between the first tag and the second probe. recapturing the second solid surface via a capillary; (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 capturing the immune complexes on a first solid surface prior to capturing the immune complexes on a first solid surface. In some embodiments, the step comprises forming the immune complex in solution. (1) is a method for capturing an immune complex on a first solid surface by attaching a first binder to the first solid surface. In some embodiments, in step (1), the immunoglobulin is pre-captured onto a surface. The complex is formed in solution and simultaneously captured on a first solid surface.

[0209] In some embodiments, the assay methods provided herein comprise detecting the immune complexes by: releasing the immune complex from the solid surface on which it is captured; ) a second probe, or (c) another nucleic acid probe that hybridizes to the first tag. and washing the additional solid surface to remove any unremoved particles. and removing the bound molecules. Including Kuru.

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

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

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

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

[0214] In some embodiments, the first tag and the second tag are are cooperatively trapped on the solid surface of

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

[0216] In some embodiments, the contiguous fragment of the first probe comprises the first fragment and the immediately adjacent fragment. wherein the first tag and the second tag are linked to form a linked nucleic acid When the junction region of the linked nucleic acid is formed, the junction region of the linked nucleic acid is complementary to the continuous fragment of the first probe. The first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the first tag or a fragment thereof, such that The piece is complementary to the second tag or a fragment thereof.

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

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

[0219] In some embodiments, the first tag and the second tag are are cooperatively trapped on the solid surface of

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

[0221] In some embodiments, the contiguous fragment of the second probe is the first fragment and the immediately adjacent a second fragment of the nucleic acid sequence, wherein 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. Thus, the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is , which is complementary to said second tag or a fragment thereof.

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

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

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

[0225] The assay methods provided herein detect an analyte in a sample, which and in step (6), detecting the immune complex.

[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; The binder binds to one molecule of the binding pair, and the second binder binds to the other molecule of the binding pair. Binds to molecules.

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

[0229] In some embodiments, in step (6): the immune complex is captured on a solid surface. In some embodiments, in step (6): the immune complex is detected simultaneously with the immobilized It is detected after being released into solution from the body surface.

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

[0231] In some embodiments, step (6) comprises linking the first tag and the second tag. The nucleic acid reporter is generated by the above method, and is composed of a fragment of the first tag and a fragment of the second tag. and detecting the nucleic acid reporter produced.

[0232] In some embodiments, step (6) comprises (a) providing surrogate nucleic acids for the first tag and the second tag (" A nucleic acid reporter is generated by linking a second surrogate ("second surrogate") to the first tag. (b) detecting a nucleic acid reporter comprising the first surrogate fragment and the second surrogate fragment; The nucleic acid reporter is formed by linking the second tag to a surrogate nucleic acid (the "first surrogate"). and generating a nucleic acid reporter comprising a fragment of the first surrogate and a fragment of the second tag. or (c) detecting the nucleic acid receptor by ligating the first surrogate with the second surrogate. a nucleic acid reader comprising a fragment of the first surrogate and a fragment of the second surrogate; detecting a surrogate; wherein the first tag or fragment thereof is a surrogate for the first surrogate; or a fragment thereof, and said second tag or fragment thereof is complementary to said second surrogate or is complementary to a fragment of

[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 a reporter for qPCR, digital PCR, or next-generation PCR. It is detected by next-generation sequencing (NGS).

[0235] In some embodiments, the nucleic acid reporter is a nucleic acid fragment that is amplified by rolling cycle amplification (RCA), strand amplification, or strand fragment amplification. Displacement Amplification (SDA), Loop-Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), or is detected by the QuantiGene assay.

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

[0237] In some embodiments, the assay methods provided herein comprise: Identify at least two analytes in a sample by simultaneously detecting related unique target IDs. Simultaneous detection is included.

[0238] In some embodiments, the assay method provided herein comprises: By adding a non-functional binder to the solution, the sigma from at least one of the analytes can be reduced. wherein the non-functional binder proportionally reduces the signal to the analyte. The first binder is conjugated to a presenting group that competes with the first binder for binding but does not bind to the first acceptor group. They are either unconjugated or conjugated.

[0239] In some embodiments, at least one analyte is a nucleic acid, and the nucleic acid analysis The first and second binders of the object 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 detecting a first target. This involves simultaneous detection of a target ID and a second target ID.

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

[0242] In some embodiments, the nucleic acid reporter formed in each sample is sample-specific. a sample ID ("sample ID"), wherein the sample ID includes: (1) a first tag or a substitute thereof; and the second tag or its substitute, or (2) in the first substitute or the second substitute or (3) the first tag or its substitute, or the second tag or its substitute. It is ligated to a substitute.

[0243] In some embodiments, the assay methods provided herein comprise: Simultaneous detection of unique sample IDs in associated nucleic acid reporters allows for the identification of at least two Simultaneous detection of analytes in a sample is included.

[0244] In some embodiments, the nucleic acid reporter comprises: (a) a first tag or a first surrogate; (b)(1) a target ID in the first tag or its surrogate, or in a second tag or second surrogate; (2) inserted between the first alternative and the second alternative, or or (3) the first tag or its substitute, or the second tag or Contains the sample ID that will be ligated to that alternative.

[0245] In some embodiments, the assay methods provided herein comprise: Simultaneous detection of the unique sample ID and the unique target ID in the nucleic acid reporter associated with each analyte. By doing so, it is possible to simultaneously detect at least two analytes in at least two samples. include.

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

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

[0248] In some embodiments, provided herein are methods for detecting an analyte in a sample. A system for forming a binder, a binder, a first presenting group, a second presenting group, , a first acceptor group, and a second acceptor group; wherein (i) the first and second binders are (ii) the first and second presentation groups each bind to a non-interfering epitope on the analyte; and The system is a system that binds to the first and second acceptor groups.

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

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

[0251] In some embodiments, the first solid surface is coupled to a first acceptor 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. include.

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

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

[0255] In some embodiments, (i) the first presenting group is a thioester group, a disulfide bond, or (ii) the second presenting group is attached to the first accepting group via a bond that is cleavable or that can be broken down; , a thioester group, a disulfide bond, or a cleavable bond to a second acceptor group or both (i) and (ii).

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

[0257] In some embodiments, provided herein are methods for detecting an analyte in a sample. A system for detecting 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”), a second nucleic acid tag (“second tag”), a first nucleic acid capture probe (“first probe”), a second nucleic acid tag (“second tag”), a second nucleic acid capture probe (“second probe”), a second nucleic acid capture probe (“second probe”), a second nucleic acid capture probe (“second probe”), a second nucleic acid capture probe (“second probe”), a second nucleic acid capture probe (“second tag ... ), and a second nucleic acid capture probe ("second probe"); wherein (i) said first and (ii) a second binder binds to a non-interfering epitope on the analyte; and (iii) the first probe or its (iii) a fragment thereof is complementary to the first tag or a fragment thereof; and (iv) a fragment thereof is complementary to the second tag or a fragment thereof. The fragment is complementary to the second tag or a fragment thereof.

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

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

[0260] In some embodiments, the first solid surface is coupled to a first probe. 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 is between 10 and 2 (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 that specifically binds to the analyte. (ii) the second binder is an antibody or antibody fragment that specifically binds to the analyte; an antibody or antibody fragment; or both (i) and (ii).

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

[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 include a second nucleic acid tag ("second In some embodiments, the second tag further comprises a second binder. is conjugated to

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

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

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

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

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

[0274] In some embodiments, the systems provided herein comprise a first solid surface and a second The method further comprises the step of: providing a solid surface on which the first solid surface is attached a first probe and an additional probe; and the second solid surface is coupled to a second probe and an additional probe. They are coupled.

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

[0276] In some embodiments, (i) the complementary fragment of the first tag and the first probe is between 10 and 2 (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 includes a binding buffer, an immobilization buffer, a wash buffer, The composition may further comprise a 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 limited to specific embodiments illustrated herein. It should be understood that the terminology used herein is not intended to be limiting. It is merely for purposes of illustrating particular embodiments and is not intended to be limiting. It should also be understood that

[0305] (5.1 Definition) Unless otherwise defined herein, scientific and technical terms used in connection with the present invention are , shall have the meaning commonly understood by a person skilled in the art. Unless required otherwise, singular terms include pluralities and plural terms include the singular. Generally, the molecular biology, immunology, genetics, and protein Nomenclature used in connection with protein and nucleic acid chemistry and hybridization, and and these techniques are well known and commonly used in the art.

[0306] As used herein, the term "detect" or its grammatical equivalents means to detect an analyte. Any means of determining the presence (i.e., whether it is present) of an analyte or any measure of an analyte The term "detecting" is used broadly to include any form of detection. or determining, measuring, or assessing the absence or amount or location of This includes quantitative, semi-quantitative, and qualitative determinations, measurements, or evaluations. If, for example, two or more different analytes are being detected in a sample, the measurement, or evaluation may be It can be relative or absolute. The term "quantifying" when used in the context of quantifying a precipitate does not mean absolute determination. Absolute quantification can refer to the amount or relative quantification of one or more control analytes of known concentration. and / or target analytes using known control analytes (e.g., by generating a standard curve). Alternatively, relative quantification can be achieved by comparing two or more different i.e., two or more analytes in order to provide a relative quantification of each of the analytes relative to one another. This can be achieved by comparing the detection levels or amounts between different target analytes.

[0307] As used herein, the term "analyte" refers to an analyte in an assay provided herein. It can be any substance (e.g., molecule) or entity to be detected by the method. The analyte is the target of the assay methods provided herein. Any biological molecule or chemical compound that needs to be purified, e.g., a peptide or protein The analyte can be a nucleic acid molecule, or a small molecule, including organic and inorganic molecules. The antigen may be a microorganism, including a bacterium or virus, or a fragment or product thereof. The analyte is one for which a specific binder can be developed and which has at least two " The binder can be any substance or entity that can simultaneously bind to the binder. In some embodiments, the analyte is a protein or polypeptide. The analytes of interest may be polypeptides, proteins, or proteins such as prions. a protein molecule, or any molecule containing a protein or polypeptide component, or In some embodiments, the analyte is a complete or partial fragment thereof. The analyte is a single molecule containing two or more molecular subunits. or complexes, which may or may not be covalently bonded to each other. and may be the same or different. The analyte that can be detected by the assay method can be a complex analyte, It can be a protein complex. Such a complex can therefore be homozygous. It can be a multimer or heteromultimer. Aggregates of molecules (e.g., proteins) can also be targeted. The aggregation analyte can be an aggregation of the same protein or different proteins. The analyte can be a protein or peptide, or DNA or RNA, etc. In some embodiments, the nucleic acid molecule may be a complex composed of the nucleic acid molecule. Precipitates are complexes composed of both proteins and nucleic acids, e.g., regulatory factors such as transcription factors. He is a child.

[0308] As used herein, the term "sample" refers to any biological and clinical sample. This can include, for example, any cell or tissue sample of an organism, or Any body fluid or preparation derived from the body, including, for example, cell cultures, cell preparations, cell lysates, Also included are environmental samples, such as soil and water samples or food samples. The sample may be freshly prepared or pre-treated in any convenient way (e.g., for storage). It can be a well-understood system.

[0309] Thus, a representative sample may include any material containing biomolecules, or, for example, food and and any other desired or target analytes, including analytes of interest, including analytes of interest, clinical and environmental samples. The sample can be a biological sample, including prokaryotic or eukaryotic cells, viruses, or other organisms. bacteriophages, mycoplasmas, protoplasts, and organelles, Such biological material may include viruses or cellular material. and non-mammalian cells, plant cells, algae including cyanobacteria, fungi, bacteria, protozoa, and the like. Representative samples 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. The sample may be pre-treated in any convenient or desired manner to prepare the sample for use in the method disclosed herein. For example, the sample can be prepared by cell lysis or can be processed by purification, isolation of analytes, etc.

[0310] As used herein, the term "conjugate" or its grammatical equivalents refers to a complex. Interaction between molecules (e.g., binder and analyte, or presenting group and accepting group) to form The interactions may be, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or phasic interactions. The interaction can be non-covalent, including von der Waals interactions. As used herein, a "binder" refers to any molecule that can bind to an analyte. In some embodiments, the binder is a molecule or entity specific for its target analyte. that is, the binder binds with a higher affinity to the other components in the sample than to the other components in the sample. In some embodiments, the binder binds to a non-target analyte. does not bind to any other compound or to a negligible or undetectable extent, or Nonspecific binding, if it occurs, is at a relatively low level that can be distinguished. The binding of the binder to the target analyte is either The binding can be distinguished from the binding of the binder to a non-target analyte. The bond between the binders is typically non-covalent. The binders used in the method are selected from those that substantially increase the binding affinity of the binder for its target analyte. covalently conjugating to a presentation group (e.g., a nucleic acid tag) without being automatically lost It is possible.

[0311] The binder can be selected to have a high binding affinity for the target analyte. In some embodiments, the binder has a viscosity of at least about 10 -4 M, at least about 10 - 6 M, or at least 10 -9 M or higher binding affinity for the target analyte. The binder must exhibit the requisite binding affinity for the target analyte. In other embodiments, the binder is a moderate or low, e.g., about 10, -4 have an affinity less than M It is possible.

[0312] The binder can be a macromolecule. In some embodiments, the binder The binder is an antibody or a binding fragment, derivative, or mimetic thereof. In this case, a heterogeneous population of antibodies with different specificities are each conjugated to the same presentation group. A polyclonal composition, such as a homogenous composition with the same specificity for the target analyte, may be used. from a monoclonal composition in which identical antibody populations are each conjugated to the same presenting group. Thus, the binder can be a monoclonal or polyclonal antibody. The antibody can be either a mAb or an antibody.

[0313] In some embodiments, the binder is an antibody fragment, derivative, or mimetic thereof. where these fragments, derivatives, and mimetics have the requisite binding to the target analyte. Such antibody fragments or derivatives typically retain the binding characteristics of the antibody of interest. To achieve this, at least V H and V L In some embodiments, In this context, the binder is an antibody fragment that binds to the analyte. refers to a molecule other than an intact antibody that contains a portion of an antibody and usually an antigen-binding site. Examples of antibody fragments Examples include Fab, Fab', F(ab')2, Fv, single-chain antibody molecules (e.g., scFv), disulfide-bonded scFv, and Fv (dsscFv), diabody, triabody, tetrabody, minibody, dual variable domain single variable domain antibodies (e.g., camelid antibodies, alpaca antibodies), heavy chain Examples include antibody single variable domains (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 this case, the binder is a single variable domain antibody.

[0314] In some embodiments, the binder is an antibody mimetic. Molecules that can specifically bind to antigens but are not structurally related to antibodies, such as Antibody mimetics are typically artificial peptides within the molar mass range of about 2-20 kDa. Nucleic acids and small molecules may also be considered antibody mimetics. Antibody mimics include affibodies, affilins, affimers, affitins, and alphabodies. Di, Anticalins, Aptamers, Avimers, DARPins, Fynomers, Kunitz domain peptides These include tides, monobodies, and nanoCLAMPs.

[0315] In some embodiments, suitable for use as binders are polynucleic acid aptamers. Polynucleic acid aptamers bind to proteins in much the same way as receptors or antibodies. It can be an RNA oligonucleotide that can act to specifically bind to See the reference by Nrad et al., Methods Enzymol. (1996), 267 (Combinatorial Chemistry), 336-367). The antibodies, fragments, derivatives, and mimetics thereof may be obtained from commercial sources and / or polyclonal antibodies. antibodies, monoclonal antibodies, fragments, derivatives and mimetics thereof, including recombinant derivatives thereof It may be prepared using any convenient technique, provided that methods for producing it are known to those skilled in the art. (See, for example, U.S. Patent Nos. 5,851,829 and 5,965,371).

[0316] In addition to antibody-based peptide / polypeptide or protein-based binding domains, The binder may be a lectin, a soluble cell surface receptor or derivative thereof, an affibody, or Phage display or ribosome display or any type of combinatorial Any combinatorially derived protein from a peptide or protein library It can also be a protein or peptide.

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

[0318] In some embodiments, the binder binds to the target analyte with the requisite affinity. Small molecules are small organic molecules. Small molecules are small molecules that can bind to target analytes. One or more functional groups necessary for structural interaction, for example, hydrophobic interaction, hydrophilic interaction, It may contain groups necessary for electrostatic or covalent interactions. In the case of proteins, small molecule binders may interact with the protein through structural interactions, e.g., water It contains functional groups necessary for elementary bonds, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and is usually At least one of amine, amide, sulfhydryl, carbonyl, hydroxyl, or carboxyl In some embodiments, at least one of the functional groups may comprise a silyl group. Small molecule binders are essentially small molecules that are capable of binding to their target analytes. modified and / or covalently linked to a presenting group (e.g., a nucleic acid tag) without adversely affecting the It may also include regions that can be involved in binding.

[0319] The small molecule binder may comprise a cyclic carbon or cyclic group substituted with one or more of the above functional groups. It may also contain heterocyclic and / or aromatic or aromatic polycyclic structures. Molecular binders include peptides, saccharides, fatty acids, steroids, purines, pyrimidines, Containing structures found among biomolecules, including 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 be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. They can also be derived from natural or synthetic compounds that can be synthesized by a number of methods. are available for random and directed synthesis of a wide variety of organic compounds and biomolecules. Libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available. Additionally, natural or synthetically produced libraries are readily available. The molecules and compounds are readily modified by conventional chemical, physical, and biochemical means. and can be used to generate combinatorial libraries. Molecules can be modified by directed or random methods, such as acylation, alkylation, esterification, amidation, etc. These small molecule binders can be subjected to chemical modifications to produce structural analogs. The term "combinatorially generated libraries of compounds" refers to compounds that are derived from libraries of natural or synthetic molecules, including biodiversity combinatorial libraries When obtained from such libraries, small molecule binders can be Demonstrates several desirable affinities for protein targets in a comprehensive binding affinity assay. is selected.

[0321] The assay methods provided herein involve first and second nucleotide sequences that bind to a non-interfering "epitope" of the analyte. The binder and the second binder are used. A pitope refers to the site on the surface of an analyte to which a binder binds. An epitope refers to the site on the surface of an analyte to which a binder binds. An epitope can be a localized region on the surface of a molecule such as an amino acid or sugar side chain. An epitope can consist of a chemically active surface grouping. An epitope is a contiguous fragment of an analyte molecule. An epitope can also be a molecule consisting of multiple non-contiguous fragments of an antigen. If the analyte is a polypeptide or protein, the epitope may be a polypeptide or protein. The sequences may include contiguous or non-contiguous sequences along the primary sequence of the peptide chain. The first binder and the second binder used in the assay method disclosed herein The first binder and the second binder are of the same molecular type. Both of the inhibitors are monoclonal antibodies that bind to non-interfering epitopes of the analyte. In some embodiments, the first binder and the second binder are different. For example, the first binder can be an antibody and the second binder can be a The compound can be a small molecule.

[0322] The term "binding moiety" when used in reference to an analyte means that the moiety as a whole binds to the analyte. Refers to a moiety, including a molecule or group of molecules, that can specifically bind to a substance The binding moiety may be one or more binders, one or more target labels, one or more sample labels, and / or one or more The binding moiety may comprise a binder, a target label, a sample label, and / or a presentation group as described above. Alternatively, the binding moiety may comprise a binder, a target label, and / or a presentation group. The molecules in the binding moiety can be linked together through covalent intermolecular interactions, non-covalent interactions, or both. by bonding intermolecular interactions or a combination of covalent or non-covalent intermolecular interactions. Thus, the molecule in the binding moiety can be attached as a moiety. binds via interaction with a molecule that is not a moiety, such as an analyte or one or more receptor groups Furthermore, the molecules in the binding moiety can be oriented by (i) intermolecular interactions between the molecules in the binding moiety. and (ii) via a molecule that is not part of the binding moiety, e.g., an analyte or one or more acceptor groups. In one embodiment, the binding moiety In some embodiments, the binding moiety comprises or consists of a binder. In some embodiments, the binding moiety comprises a presentation group. In other embodiments, the binding moiety comprises or consists of a sample label. In one embodiment, the binding moiety comprises or comprises a binder and a target label. In some embodiments, the binding moiety comprises or comprises a binder and a presenting group. In some embodiments, the binding moiety comprises a binder and a sample label. In a further embodiment, the binding moiety comprises 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 presenting group and a sample label. In yet another embodiment, the binding moiety comprises or consists of a binder, a target In some embodiments, the binding moiety comprises or consists of a label, and a presentation group. The label comprises or consists of a binder, a target label, and a sample label. wherein the binding moiety comprises or consists of a binder, a presentation group, and a sample label. In some embodiments, the binding moiety comprises or comprises a target label, a presenting group, and a sample label. In other embodiments, the binding moiety is a binder, a target label, a presentation group, or a combination thereof. and a sample label. In some embodiments, the binding moiety , a binder, a target label, a presentation group, and a sample label. In some embodiments, the binding moiety comprises any combination or permutation of binders. -, a target label, a presentation group, and a sample label, or any two of these. In some embodiments, the binding moiety is a binder, target, or antibody, in any combination or permutation. It comprises or consists of any three of a label, a presenting group, and a sample label. In some embodiments, the binding moiety comprises a binder, a target label, a presentation group, and a sample label. It consists of or includes all four of these.

[0323] The terms "presenting group" and "accepting group" are used in conjunction with each other herein, The term refers to a binding pair that can form a complex under appropriate conditions. When used in an assay method, the presenting group binds to a binder of the target analyte. The acceptor 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." which allows the captured binder to be released from the solid surface. In embodiments, the bond formed between the presenting group and the accepting group is "renewable" and This allows the carboxyl group to be recaptured on another solid surface coupled to the same acceptor group. Similar to the discussed binding pair of a "binder" and its "analyte," a "presenting group" and an "accepting group" The binding pair can take various forms. Examples of binding pairs of "presenting group" and "accepting group" include These include antigens and antibodies to the antigens (including fragments, derivatives, or mimetics thereof), ligands and their Receptor, complementary strand of nucleic acid, biotin and avidin (or streptavidin or neutra These include, but are not limited to, lectins and carbohydrates (and vice versa). Further binding pairs of "presenting group" and "accepting group" include fluorescein and antifluorescein. Digoxigenin / anti-digoxigenin, and DNP (dinitrophenol) / anti-DNP (inverse case) In some embodiments, the "presenting group" and "acceptor group" binding pair is and complementary strands of nucleic acid, referred to as "tags" and "probes." In this case, the binding pair of the "presenting group" and the "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 "label" refers to a moiety that facilitates detection and identification of the sample source of a target. Suitable labels for labeling the target and sample include those that can be associated with a particular target or sample. In the context of the present disclosure, target labels and labels capable of providing an identifier are also included. General labels that can be used for sample labeling and / or targeting via sequencing or a sequence of nucleotides that can be associated with the sample. In some embodiments, the target label comprises a target ID. In some embodiments, the target label consists of a target ID. In other embodiments, the target label is a target ID. The sample label includes a sample ID. In some embodiments, the sample label consists of a sample ID. In an embodiment, the sample label is a sample ID. Other labels include molecules that contain other distinguishable or correlated information, such as fluorescent molecules. A combination or arrangement of molecules or fluorescent molecules, and / or a colorimetric moiety or combination of colorimetric moieties. Other labels contemplated for the present disclosure include luminescent, photochromic, and Examples include scattering agents, radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, and magnetic particles. Patents that teach the use of such labels include U.S. Patent Nos. 3,817,837; Nos. 850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,24 Many labels are commercially available and suitable for use in connection with the present invention. can be done.

[0325] "Identification barcode" or "ID" when used in relation to a target or sample The term refers to the direct identification of a target or sample by the identifying information contained in a molecule or series of molecules. It refers to a molecule or set of molecules that can be used to identify or indirectly identify a specific molecule. A suitable ID is one in which the molecule or combination of molecules used as the ID differentiates a particular target or sample from another target or sample. Identifying or otherwise distinguishing from a sample and associating with an intended target or sample To the extent possible, a given sequence, a unique fluorescent label, a unique colorimetric label, a sequence of fluorescent labels, a colorimetric label It can be a nucleic acid molecule having the sequence of any of the above, or any other molecule or combination of molecules. Such nucleic acid molecules used as IDs are also known as barcode sequences. Such IDs may be used to identify specific targets or samples that are not directly related to other targets or samples. Identifying or otherwise distinguishing from a sample and associating with an intended target or sample To the extent possible, further variants containing information derived from, but not identical to, the original ID It can also be a biomolecule. For example, a nucleic acid ID can be a sequence of the original nucleic acid barcode and / or a The reverse complement of the nucleic acid barcode sequence may be included, since both are intended to be This is because it allows identification and association with the target or sample being depicted. The sequence may be a sequence of an intended sample, an intended target, or any portion of an intended sample or target. Any natural or non-natural sequence that would not exist unless introduced as a barcode sequence into a molecule. The barcode sequence can then be used to distinguish and associate with a 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 in the population has a barcode that is different from all other nucleic acid probes in the population. Alternatively, each nucleic acid probe in the population may contain a sequence of several nucleic acid probes in the population. The barcode sequence may be different from most or all other nucleic acid probes. For example, all reporters generated from immune complexes derived from one sample are generated using the same test compound. In another example, multiple immunogens from the same sample can have the same sample barcode sequence (sample ID). Every reporter generated from an epigenetic complex has a different target barcode sequence (target ID). Furthermore, it is possible to detect multiple antibodies from the same sample, against the same target, and with the same binder. All reporters generated from the same immunoconjugate have the same target barcode sequence (target ID). It is possible.

[0326] The term "and / or" as used in phrases such as "A and / or B" herein means A and B; A or B; A (only); and B (only). Similarly, "A, B, and The term "and / or" as used in phrases such as "and / or C" refers to any of the following embodiments: A, B, C, and / or C. 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 are nucleic acid-based immunoassays that address the limitations of existing immunoassays and The present invention provides an assay method that allows single molecule detection of immune complexes by signal amplification. The method provided in reduces background signal by a capture and release mechanism. Thus, provided herein are methods for detecting analytes in a sample, including capture and release mechanisms. 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 utilize a capture and release mechanism to eliminate nonspecific background Reduces round signal. Captures immune complexes on a solid surface and releases them into solution. The process of returning to can be applied to various assay formats disclosed herein ( For example, Figures 4A-4D and 8A-8D).

[0329] 5.2.1.1 Capture and Release Using Two Capture Binders In some embodiments, the present invention provides a method for treating a vascular endothelial cell, such as a vascular endothelial cell, comprising administering to a subject a vascular endothelial cell, a vascular endothelial cell, or ... as provided herein, as shown in Figures 4A, 8A, and 8B. The assay method provided involves capturing two receptor groups on two solid surfaces, respectively. Some embodiments use a capture and release mechanism involving two binders that can Provided herein is an assay method for detecting an analyte in a sample, comprising: The process involves the following steps: (1) mixing a first binder, a second binder, and the sample in a solution, the first and second binders bind to non-interfering epitopes on the analyte; and and forming a complex, the immunocomplex comprising a first presenting group conjugated to the first binder. and a first accepting group coupled to the first surface. captured on a solid surface); (2) washing the first solid surface to remove unbound molecules; (3) disrupting the bond between the first presenting group and the first accepting group, thereby from said first solid surface; (4) introducing a second solid surface and a second display conjugated to the second binder; and a second acceptor group coupled to the second solid surface. recapturing the coalescence; (5) washing the second solid surface to remove unbound molecules; and (6) detecting the immune complex. The assay method includes:

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

[0331] As one skilled in the art will appreciate, additional rounds of capture / release can be used to reduce non-specific background. In some embodiments, binding 1 may further reduce the sound signal. At least one additional round of capture / release can be performed via Binder 1. Specifically, the immune complex released from surface 2 can be bound to PG on binder 1. By forming another bond between 1 and RG1 on new surface 1, the RG1 is recaptured by new surface 1. In some embodiments, bond 2 is renewable and can be at least Also, one additional round of capture / release can be performed via Binder 2. Specifically, immune complexes released from either Surface 1 or Surface 2 bind to PG2 on Binder 2. and RG2 on new surface 2, forming another bond between them. 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 can be bound by a single capture / release cycle. Only cycles are performed.

[0332] Thus, in some embodiments, the assay methods provided herein involve immunoassay. releasing the immune complex from the solid surface on which it is captured; and recapturing the compound on a further solid surface coupled to the group, and and washing to remove unbound molecules. In some embodiments, the method further comprises a recapture cycle. The assay method involves liberating the immune complex from the solid surface on which it is captured, recapturing the aggregate on a further solid surface coupled to a second acceptor group; and and washing the solid surface to remove unbound molecules. The method further includes at least one additional recapture cycle. can.

[0333] Releasable bonds can be used in many different approaches known to those skilled in the art of protein immobilization. For example, in some embodiments, the releasable bond is In some cases, the attachment is via a thioester group (e.g., U.S. Pat. No. 4,284,553). In embodiments, the releasable bond is a cleavable bond (see, e.g., Leriche, Bi Organic & Med. Chem. 20(2): 571-581(2012)). In some embodiments, the free radicals A suitable bond is a disulfide bond (see, 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 Linkages include, for example, phosphodiester, phospholipid, ester, or β-galactose. It is a bond that can be cleaved with an appropriate enzymatic activity. The releasable bond can be catalyzed by a chemoenzymatic reaction such as the Staphy-eSrtA pair (see, e.g., Ham et al., Nature Com communications 7:11140(2016)) and others (Rabuka, Curr. Opin. Chem. Biol. 14, 7 90-796(2010); Rashidian, J. Am. Chem. Soc. 134:8455-8467(2012)); Kosa, It is a bond that can be cleaved by the following method (Nat. Methods 9, 981-984 (2012)). In some embodiments, the releasable bond is a bond between an arginine residue and a 4-(oxoacetyl)phenyl Adsorbents derivatized with hydroxyacetic acid (see, e.g., Duerksen-Hughes, Biochemistry, 28 (21):8530-6(1989)). In some embodiments, the releasable bond is , disrupted by binding competition (e.g., Nguyen, Biomol. Eng. 22 (2005) 147-150). Renewable bonds are also known by those skilled in the art of protein immobilization. This can be achieved by a number of different approaches, for example, by using binding pairs (e.g., antigens) Non-covalent bonds, including hydrogen bonds, formed between a molecule (e.g., a molecule and an antibody, a ligand and a receptor, or a complementary nucleic acid) In this case, the releasable and regenerative bond can be, for example, a metal affinity bond. (e.g., Cheung, Appl. Microbiol. Biotechnol. 96, 1411-1420 (2012)), N- lamin structures (e.g., Hui, Biomacromolecules 14 585-601 (2013)), or disulfides By using a methylated bond (e.g., Boitieux, Anal. Chim. Acta 197: 229-237 (1987)), This can also be achieved.

[0334] In some embodiments, in step (3), the immune complex is lysed by lysing an excess of free first By adding either the presenting group or the free first acceptor group to the solution, binding competition can be achieved. The "free" presenting group is then conjugated to a binder. A "free" acceptor group refers to a presenting group that is not coupled to a solid surface. refers to a suitable acceptor group.

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

[0336] In some embodiments, 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"), wherein the The first probe or a fragment thereof is complementary to the first tag or a fragment thereof. In an embodiment, the second presentation group is a second nucleic acid tag ("second tag") and is a second receptor. The capture group is a second nucleic acid capture probe ("second probe"), wherein the second probe The tag or fragment thereof is complementary to the second tag or fragment thereof.

[0337] In some embodiments, as shown in FIG. 4B, the first presentation group comprises a first tag. and the first acceptor group is a first probe, wherein the first probe or its The fragment is complementary to the first tag or a fragment thereof; and the second presentation group is a second tag. and the second acceptor group is a second probe, wherein the second probe or a fragment thereof is , complementary to the second tag or a fragment thereof. 1. 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 a solution, the first and second binders bind to non-interfering epitopes on the analyte; and and forming a complex, wherein the immunocomplex is conjugated to the first binder. Hybridization between a first tag and a first probe coupled to the first surface. via ion onto a first solid surface in contact with the solution; (2) washing the first solid surface to remove unbound molecules; (3) The immune complex is subjected to hybridization between the first tag and the first probe. from the first solid surface via dissociation of (4) introducing a second solid surface and a second tag conjugated to the second binder; and a second probe coupled to the second solid surface. recapturing the immune complexes onto the second solid surface via (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 comprise a method for detecting a second tag and a first tag. It captures the immune complex through dissociation of the hybridization between the two probes The immunoconjugate is then released from the solid surface on which it is coupled to the first acceptor group. and washing the additional solid surface to remove unbound molecules. and further performing at least one additional recapture cycle between steps (5) and (6), comprising: In some embodiments, the assay methods provided herein comprise a first tag and the first probe, through the dissociation of the hybridization between the immune complex, Releasing the immunoconjugate from the solid surface where it is captured and coupling the immunoconjugate with a second acceptor group. and recapturing the unbound ATP on a further solid surface, and washing the further solid surface. at least one additional recapture cycle between steps (5) and (6), comprising: Further includes:

[0339] 5.2.1.2 Capture and Release Using One Capture Binder In some embodiments, the present invention provides a method for treating a vascular endothelial cell, such as a vascular endothelial cell, comprising administering to a subject a vascular endothelial cell, a vascular endothelial cell, or ... as provided herein, as shown in Figures 4C, 8C, and 8D. The assay method provided uses two binders to form an immune complex. , one binder captured by a solid surface via one releasable and renewable bond In some embodiments, a capture and release mechanism involving only the first binder is used. In some embodiments, the antibody is captured by two solid surfaces in succession. Provided herein is an assay method for detecting an analyte in a sample, comprising: Process: (1) mixing a first binder, a second binder, and the sample in a solution; The first and second binders bind to non-interfering epitopes on the analyte to form an immune complex. and wherein the immunocomplex is conjugated to the first binder. via a bond between a first presenting group and a first accepting group coupled to the first solid surface , captured on a first solid surface in contact with the solution); (2) washing the first solid surface to remove unbound molecules; (3) disrupting the bond between the first presenting group and the first accepting group, thereby from said first solid surface; (4) introducing the first presenting group onto a second solid surface and coupling the first presenting group to the second solid surface; recapturing the immune complex via binding between the second receptor group; (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. In some embodiments, the second accepting group is the same as the first accepting group.

[0341] As shown in Figures 4C, 8C, and 8D, the capture / release is determined by the first binder ("binder"). a first presenting group ("PG1") on a first solid surface ("Surface 1") and a first accepting group ("RG1") on a first solid surface ("Surface 1"); This is achieved through a releasable and renewable bond between the first one ("bond 1") and the second one ("bond 2"). The same presenting group (PG1) can then be attached to a second accepting group (PG2) on a second solid surface ("Surface 2"). A second bond ("bond 2") can be formed with RG1 ("RG2"), where RG2 is the same as RG1. In some embodiments, bond 2 is also releasable and the immune complex is It can be detected either on the surface 2 or after release from the surface 2. In an embodiment, Bond 2 is not releasable and the immune complexes can be detected on Surface 2. can.

[0342] As one skilled in the art will appreciate, additional rounds of capture / release can be used to reduce non-specific background. Since bond 1 is renewable, at least 1 Two additional rounds of capture / release can be performed via Binder 1. In other words, immune complexes released from surface 2 bind between PG1 on binder 1 and RG1 on new surface 1. By forming another bond with the surface, it can be recaptured by a new surface. In some embodiments, Bond 2 is also renewable and can be used to bind immunoglobulins released from Surface 1 or Surface 2. The complex is formed by forming another bond between PG1 on binder 1 and RG2 on new surface 2. and can be recaptured by a new surface 2. Both Bond 1 and Bond 2 are renewable, and multiple recapture cycles can be performed on Bond 1, Bond 2, or both. This can be done through the following methods.

[0343] Thus, in some embodiments, the assay methods provided herein involve immunoassay. releasing the immune complex from the solid surface on which it is captured; and recapturing the compound on a further solid surface coupled to the group, and and washing to remove unbound molecules. In some embodiments, the method further comprises a recapture cycle. The assay method involves liberating the immune complex from the solid surface on which it is captured, recapturing the aggregate onto a further solid surface coupled to a second acceptor group; Further washing the solid surface to remove unbound molecules. Further comprising at least one additional recapture cycle. This can be done.

[0344] As noted above, releasable and regenerative bonds include those disclosed herein. This can be achieved by many different approaches known to those skilled in the art of protein immobilization, including In some embodiments, the releasable and regenerative bond can be a nucleic acid hybrid. are formed through hybridization, where the presenting group and the accepting group are complementary to each other. Contains nucleic acids.

[0345] In some embodiments, the first presentation group is a nucleic acid tag (“first tag”). One acceptor group is a nucleic acid capture probe ("first probe"), where the first probe The first tag or a fragment thereof is complementary to the first tag or a fragment thereof. As discussed above, provided herein are assays for detecting an analyte in a sample. 10. A method comprising: (1) mixing a first binder, a second binder, and the sample in a solution; The first and second binders bind to non-interfering epitopes on the analyte to form an immune complex. and wherein the immunocomplex is conjugated to the first binder. A first nucleic acid tag ("first tag") and a first nucleic acid capture molecule coupled to the first solid surface. The first probe contacts the solution through hybridization between the capture probe ("first probe"). captured on a solid surface); (2) washing the first solid surface to remove unbound molecules; (3) The immune complex is subjected to hybridization between the first tag and the first probe. from the first solid surface via dissociation of (4) a second solid surface coupled with a second nucleic acid probe ("second probe"); and the immune complex is introduced into the first tag and the second probe by hybridization. recapturing the second solid surface via (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 comprise detecting the immune complexes by: releasing the immune complex from the solid surface on which it is captured; The probe may be either the first tag or another nucleic acid probe that hybridizes to the first tag. recapture onto a further solid surface coupled with a probe capable of being captured; and a step between steps (5) and (6), comprising: washing the solid surface comprising the compound to remove unbound molecules. Further includes at least one additional recapture cycle.

[0348] At least the conditions for nucleic acid hybridization are completely different from those for protein binding. The use of nucleic acid hybridization as a capture / release mechanism is disclosed herein. This provides several advantages to the assay method. First, the first binder and the second binder Binding to target analytes by the binders allows for fast binding kinetics and large sample inputs. Second, nucleic acid hybridization can be performed in a solution that allows Capture of immune complexes onto a solid surface using a fluorophore is generally more efficient, specific, and predictable. The sequence of the capture probe should be designed to specifically target the intended immune complex. This is useful in multiplexed assay formats. The dissociation of hybridization as a mechanism is achieved by changing the salt concentration in the buffer. This maximizes the efficiency of release while maintaining the integrity of the immune complex. capture probes by either shorter segments or A,T-rich sequences. By intentionally designing a relatively weak hybridization bond between the The designated hybridization bond dissociates, releasing the immune complex and simultaneously Other hybridizations between the immunocomplex and longer or stronger complementary sequences are stabilized. Fourth, it is possible to identify suitable release buffer conditions that maintain the hybridization state. Capture / release by lysis can be resumed and repeated without loss of efficiency and selectivity. Multiple cycles can be performed. Thus, additional rounds of capture / release can be performed at the desired level. The assay can be run until a low background of 1000 nm is reached. The release mechanism ensures unprecedented predictability and reliability of the assay method. An unexpected benefit of the present invention is that the nucleic acid capture probes coupled to the first and second surfaces The negatively charged oligonucleotide tag causes the first binder and the second binder. This helps to further reduce non-specific inclusion of binders.

[0349] 5.2.2 Immune Complex Formation The assay method disclosed herein comprises the steps of: (1) detecting a first binder and a second binder; and a sample in solution to form an immune complex, wherein the first and The second binder binds to a non-interfering epitope on the analyte, and the immune complex binds to the first a first presentation group conjugated to a first binder and coupled to a first surface; Through the bond between the first acceptor groups, the first solid surface is captured in contact with the solution. In some embodiments, the first presenting group is a nucleic acid tag, and the first accepting group is a nucleic acid capture protocol. a 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 assay method is It can be any molecule or part of a molecule that binds to a specific target analyte. Binders include any protein, peptide, nucleic acid, carbohydrate, lipid, or small molecule. In some embodiments, the binder comprises an antibody. In some embodiments, the binder comprises an antibody fragment. In some embodiments, the binder comprises a small molecule. nothing.

[0351] The binders used in the assay methods disclosed herein may be those that contain a presenting group (e.g. The binder and the presenting group can be conjugated to a nucleic acid tag. They can be attached either directly or indirectly through a linking group. When utilized, such groups are designed to provide covalent attachment of the presenting group to the binder. and to maintain the desired binding affinity of the binder for its target analyte. The linking group can vary depending on the binder. In this case, the linking group is usually biologically inert. A variety of linking groups are known to those skilled in the art, and It can be used in the assay methods disclosed herein. wherein the linking group has a reactive functional group that can be covalently bonded to a presenting group or binder. The amine-containing amine comprises a spacer group terminating at either end.

[0352] Spacer groups include aliphatic and unsaturated hydrocarbon chains, oxygen (such as polyethylene glycol), Spacers containing heteroatoms such as ethers) or nitrogen (polyamines), peptides, Spacer groups may include hydrates, cyclic or acyclic systems which may contain heteroatoms. is a metal ion that binds to a metal in such a way that it coordinates two or more ligands to form a complex. Specific spacer moieties include: 1,4-diaminohexyl , xylylenediamine, terephthalic acid, 3,6-dioxaoctane diacid, ethylenediamine -N,N-diacetic acid, 1,1'-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid), 4,4'-ethylenedi Potentially reactive functional groups include nucleophilic functional groups (amines, alkane, etc.). chol, thiol, hydrazide), electrophilic functional groups (aldehyde, ester, vinyl, keto functional groups capable of cycloaddition reactions, disulfides, epoxides, isocyanates, and maleimides Specific examples include primary and secondary Amines, hydroxamic acids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl esters Carbonyl imidazole, nitrophenyl ester, thiamin trifluoroethyl ester, glycidyl ether, vinyl sulfone, and maleimide. Specific linker groups that may be used herein include heterofunctional compounds, e.g. For example, azidobenzoylhydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pi Lysyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyl Adipimidate, Disuccinimidyl Tartrate, N-Maleimidobutyryloxysuccinimide Succinimidyl ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-sulfosuccinimidyl Succinimidyl [4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl [ 4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl-4- [N-Maleimidomethyl]cyclohexane-1-carboxylate, 3-(2-pyridyldithio)propanol Pionic acid N-hydroxysuccinimide ester (SPDP), 4-(N-maleimidomethyl)-cyclopentasiloxane Hexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC) and the like are also included.

[0353] Binder / presenting group conjugates utilized in the assay methods disclosed herein The esters can be prepared using any method known in the art. In embodiments, the presenting group (e.g., nucleic acid tag) is attached to the nucleic acid, either directly or via a linking group. , can be conjugated to a binder. The components are known in the art. can be covalently bonded to each other via functional groups, such as Such functional groups can be attached to the structure by one or more steps, such as oxidation, reduction, cleavage, etc. It can be present on a component or can be introduced onto the component. Functional groups that can be used to covalently bond the molecule include: hydroxy, sulfhydryl, and Various components that are modified to provide covalent bonds include alkyl, aryl, and amino. The specific moiety does not substantially adversely interfere with the desired binding affinity of that component for the target analyte. If necessary and / or desired, the components may be selected not to be harmful. Certain portions of may be protected with blocking groups, as known in the art. See, for example, Green & Wuts, Protective Groups in Organic Synthesis, See, e.g., Electron Microscopy Synthesis (John Wley & Sons) (1991); U.S. Patent No. 5,733,523.

[0354] When a nucleic acid tag is used as the presenting group, the binder / presenting group conjugate is a nucleic acid- They can also be produced using in vitro protocols that result in protein conjugates. Examples of such in vitro protocols of interest include: RepA-based protocols ( See, for example, Fitzgerald, Drug Discov. Today (2000) 5:253-258 and WO 98 / 37186. ), ribosome display-based protocols (see, e.g., Hanes et al., Proc. Nat. 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: 1 19-35; and WO 98 / 54312).

[0355] When used in the assay methods disclosed herein, an acceptor group (e.g., a nucleic acid capture The capture probe may be directly or, for example, using a probe as described above for linking the presenting group and the binder. by any means known in the art, which may be either indirectly via a linking group such as For example, the acceptor group can be "coupled" to a solid surface by a covalent linkage ( by chemical cross-linking) or by non-covalent association, e.g., streptavidin-biotin. tin-based coupling (biotin is provided on one domain and streptavidin is provided on the other) provided in another domain) can be coupled to a solid surface. In some embodiments, for example, the solid surface is a carboxyl group, e.g., a carbonyl group on a magnetic bead. The nucleic acid capture probe acts as a presenting group and couples with the nucleic acid capture probe via the bond between the acid and the nucleic acid capture probe. In some embodiments, the solid surface is a surface of a magnetic bead that can be coated with the magnetic material. can be covalently coupled to Protein A / G as a presentation group.

[0356] The presenting and accepting groups may be any of the groups disclosed herein or otherwise known in the art. It can be a conjugate, including an antigen and an antibody (fragment, derivative, or mimic thereof) to the antigen. mimetics), ligands and their receptors, complementary strands of nucleic acids, biotin and avidin (or streptavidin), avidin or neutravidin), lectins and carbohydrates (and vice versa) Further examples of binding pairs of "presenting groups" and "accepting groups" include, but are not limited to, fluoro groups. fluorescein and antifluorescein, digoxigenin / antidigoxigenin, and DNP (dinitrofen In some embodiments, the "presentation" may be The binding pairs of "group" and "acceptor group" are complementary strands of nucleic acid and are called "tag" and "probe." In some embodiments, the binding pair of a "presenting group" and an "acceptor group" is an antigen and an antibody, or are antigens and antibody fragments.

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

[0358] The solid surface may be any surface known in the art that can be used for immobilizing molecules. In some embodiments, the solid surface is a support to which nucleic acids are attached. It can be any surface that is suitable for attachment and that facilitates the assay process. Examples of solid surfaces include beads (e.g., magnetic beads, xMAP® beads), particles, Colloids, single surfaces, tubes, chips, multi-well plates, microtiter plates Exemplary solids include plates, slides, membranes, cuvettes, gels, and resins. Surfaces can include the surfaces of magnetic particles and the wells of a microtiter plate. When the solid phase is a particulate material (e.g., beads), it can be placed in the wells of a multiwell plate. In some embodiments, the cells can be distributed among the wells to allow for parallel processing. The solid surface is the surface of a magnetic bead. The magnetic bead can be coupled to a presentation group. In some embodiments, the magnetic beads are carboxylate-modified magnetic beads. Magnetic beads, amine-blocked magnetic beads, oligo(dT)-coated magnetic beads, Streptavidin-coated magnetic beads, Protein A / G-coated magnetic beads These can be magnetic beads or silica-coated magnetic beads. In an embodiment, 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. What are the first and second solid surfaces used in the assay methods disclosed herein? In some embodiments, the magnetic particles disclosed herein are Both the first and second solid surfaces used in the assay method are microtiter plates. The surface of the interplate.

[0359] As noted above, the analyte measured in the assay methods disclosed herein can be any In some embodiments, the analyte can be a protein or a biological molecule. 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 comprises at least one protein and at least one nuclear In some embodiments, the analyte is a nucleic acid analyte. be.

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

[0361] In some embodiments, the analyte is a binding pair of two different proteins, One binder binds to one protein of the binding pair, and the second binder binds to the other protein of the binding pair. Thus, provided herein are methods for detecting a target protein in a sample. 1. An assay method for detecting protein-protein interactions, 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 binds to another protein of the binding pair to form an immune complex, and The coupling occurs when a first presentation group conjugated to the first binder is coupled to a first surface. The first solid surface is in contact with the solution via a bond between the first acceptor group and the first bound to the first solid surface. be); (2) washing the first solid surface to remove unbound molecules; (3) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (4) introducing a second solid surface and a second presentation group conjugated to the second binder; and a second acceptor group coupled to the second solid surface. recapturing the body; (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 presentation group is a first nucleic acid tag (“first tag”). and the first acceptor group is a first nucleic acid capture probe ("first probe"), wherein the The first probe or a fragment thereof is complementary to the first tag or a fragment thereof. In an embodiment, the second presentation group is a second nucleic acid tag ("second tag") and is a second receptor. The capture group is a second nucleic acid capture probe ("second probe"), wherein the second probe The tag or fragment thereof is complementary to the second tag or fragment thereof.

[0363] In some embodiments, provided herein are methods for detecting protein-protein interactions in a sample. 1. An assay method for detecting protein interactions, 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 the antigen-binding protein binds to the other protein of the binding pair to form an immune complex; and The immunoconjugate comprises a first surface presenting group conjugated to the first binder. the first solid surface in contact with the solution via a bond between a first accepting group coupled to captured by); (2) washing the first solid surface to remove unbound molecules; (3) disrupting the bond between the first presenting group and the first accepting group, thereby from said first solid surface; (4) introducing the first presenting group to a second solid surface coupled with a second accepting group; and recapturing the immune complex to the second solid surface via binding between the antibody and the second acceptor group. and; (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 presentation group is a first nucleic acid tag (“first tag”). and the first acceptor group is a first nucleic acid capture probe ("first probe"), wherein the The probe or a fragment thereof is complementary to the tag or a fragment thereof. wherein the second acceptor group is a second nucleic acid capture probe ("second probe"), The probe or a fragment thereof is complementary to the tag or a fragment thereof.

[0365] The analytes can be nucleic acid molecules (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 allow nucleic acid molecules to be isolated from plasma and It can be detected directly in samples such as urine. The entity is 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, The present invention relates to an assay method for detecting an analyte in a nucleic acid sample, the method comprising the steps of: (1) mixing a first binder, a second binder, and the sample in a solution, The first and second binders bind to non-interfering epitopes on the nucleic acid analyte to form an immune complex. and forming a complex, wherein the immunocomplex is conjugated to the first binder. via a bond between a first presenting group and a first accepting group coupled to the first surface, a first solid surface in contact with the solution; wherein the first and second binding sites of the nucleic acid analyte are captured on the first solid surface; the nucleotide sequence is a nucleic acid that is complementary to a fragment of the nucleic acid analyte; (2) washing the first solid surface to remove unbound molecules; (3) disrupting the bond between the first presenting group and the first accepting group, thereby from said first solid surface; (4) introducing a second solid surface and a second display conjugated to the second binder; and a second acceptor group coupled to the second solid surface. recapturing the coalescence; (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 are methods for detecting an analyte in a nucleic acid sample. 1. An assay method for detecting a marker comprising the steps of: (1) A first nucleic acid tag ("first tag"), a second nucleic acid tag ("second tag"), and the sample. mixing in a solution (wherein the fragment of the first tag and the fragment of the second tag are each complementary to various fragments of the nucleic acid analyte and forming immune complexes; wherein the immune complex comprises a first tag and a first antibody coupled to the first solid surface. The solution is then bound to the nucleic acid capture probe ("first probe") via hybridization between the nucleic acid capture probe and the first probe. the first probe or fragment thereof is captured on a first solid surface in contact with the liquid, complementary to one tag or a fragment thereof); (2) washing the first solid surface to remove unbound molecules; (3) The immune complex is subjected to hybridization between the first tag and the first probe. from the first solid surface via dissociation of (4) introducing the immunocomplex to a second solid surface, and allowing the immunocomplex to bind to the second tag and the second solid surface; Hybridization between a second nucleic acid capture probe ("second probe") coupled to the recapture to said second solid surface via cleavage; (wherein said second probe or the fragment of which 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 includes analysis of mutations, methylations, translocations, fusions, and / or copy number variations. For high-throughput analysis of multiple targets by next-generation sequencing (NGS) Alternatively, the selected targets can be analyzed using qPCR, digital PCR, or other nucleic acid analysis techniques. It can be analyzed by

[0368] Step 1 of the assay methods provided herein can be performed in a variety of different approaches. For example, in some embodiments, the first binder can be contacted with the sample. The analyte can be pre-bound to a first surface that captures the analyte when the analyte is introduced. In some embodiments, the second binder is added after the sample or simultaneously with the sample, allowing multiple additions. The addition / incubation / washing steps allow immune complexes to form on the surface. do.

[0369] Thus, in some embodiments, step (1) comprises attaching the immune complex to a first solid surface. In some embodiments, the method comprises forming the immune complex in solution before capturing it on a surface of the antibody. In the method, step (1) comprises applying a first binder to the first solid surface before capturing the immune complex on the first solid surface. In some embodiments, step (1) comprises pre-capturing the antibody on the first solid surface. In this method, immune complexes are formed in solution and simultaneously captured on a first solid surface.

[0370] In some embodiments, the first binder is pre-bound to the first surface and the sample is solution to allow the analyte to bind to the first binder, and then a second binder is added to the The binder is added to the antibody to bind to the analyte, forming an immune complex consisting of the analyte and the binder. In some embodiments, the first binder is pre-bound to the first surface and the sample is and a second binder are added simultaneously to form an immune complex. Various approaches or different orders of addition / incubation / washing as disclosed in the specification may be used. It will be understood that variations having the same structure can be used to form immunoconjugates. .

[0371] (5.2.3 Alternative capture probe configurations) As noted above, provided herein are assays for detecting an analyte in a sample. 1. A method comprising the steps of: (1) mixing a first binder, a second binder, and the sample in a solution, the first and second binders bind to non-interfering epitopes on the analyte; and and forming a complex, wherein the immunocomplex is conjugated to the first binder. Hybridization between a first tag and a first probe coupled to the first surface. via a filtration onto a first solid surface in contact with the solution; (2) washing the first solid surface to remove unbound molecules; (3) The immune complex is subjected to hybridization between the first tag and the first probe. from the first solid surface via dissociation of (4) a second tag introduced onto a second solid surface and conjugated to a second binder; the immunocomplex via binding between a second probe coupled to the second solid surface. recapturing the body; (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 a solution; The first and second binders bind to non-interfering epitopes on the analyte to form an immune complex. and wherein the immunocomplex is conjugated to the first binder. A first nucleic acid tag ("first tag") and a first nucleic acid capture molecule coupled to the first solid surface. The capture probe ("first probe") contacts the solution via hybridization between the capture probe and the first probe. (c) being captured on a first solid surface; (2) washing the first solid surface to remove unbound molecules; (3) The immune complex is subjected to hybridization between the first tag and the first probe. from the first solid surface via dissociation of (4) a second solid surface coupled with a second nucleic acid probe ("second probe"); and the immune complex is introduced into the first tag and the second probe by hybridization. recapturing the second solid surface via (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 configuration shown in Figures 4A-4D and 6A, Figure 6B shows a configuration in which the capture probe is Two segments: one complementary to the tag on the binder and another capturing protein linked to the surface. This shows an example of indirect capture, where one probe contains another that is complementary to the other. The advantage of this technique is that, for example, the sequence on the capture probe that is directly coupled to the solid surface is commonly can be ubiquitous (e.g., polyT or A), thus enabling multiplexed assays Figure 6C shows a surface where the capture probe is biotinylated and the surface is Another exemplary approach for indirect capture is to coat the antibody with streptavidin or avidin. Therefore, the capture probe is biotin-streptavidin / avidin. The antibody is coupled to the surface by highly specific binding of the antibody.

[0374] Thus, in some embodiments, direct capture is achieved by capturing a first probe onto a first solid support. Directly coupled to a body surface for use in the assay methods provided herein In some embodiments, the second probe is directly attached to the second solid surface (FIG. 6A). In some embodiments, the first probe is coupled to a first solid The first probe is coupled directly to a surface, and the second probe is coupled directly to a second solid surface. It will be ringed.

[0375] In some embodiments, indirect capture occurs when the first probe directly contacts the first solid surface. hybridizes to a universal probe that is selectively coupled to the first probe, The assay method provided herein, wherein the fragment of the universal probe is complementary to the universal probe or a fragment thereof. In some embodiments, the second probe is used in a second hybridized to a universal probe directly coupled to a solid surface, wherein the The fragment of the second probe is complementary to the universal probe or a fragment thereof. In embodiments, the first probe is a universal probe that is directly coupled to the first solid surface. The probe hybridizes to the second probe, and the second probe is coupled directly to a second solid surface. The hybridization occurs with a universal probe that is tagged with the target gene.

[0376] In some embodiments, indirect capture involves attaching the first probe directly to the first solid surface. Conjugates with biotin that binds to either streptavidin or avidin that is selectively coupled. Gated and used in the assay methods provided herein (Figure 6C). In one embodiment, the second probe is directly coupled to a second solid surface. Conjugated with biotin, which binds to streptavidin or avidin. In one embodiment, the first probe is directly coupled to the first solid surface. A second protease conjugated to biotin that binds to streptavidin or avidin. The probe is a streptavidin or avidin probe that is directly coupled to a second solid surface. It is conjugated with biotin, which binds to

[0377] In some embodiments, cooperative capture is performed in the assay methods described herein. As shown in Figure 6D, the capture probe can be used with binder 1 and The assay conditions are such that the complementary strands of the nucleic acid tag simultaneously hybridize to the shorter segments of the two nucleic acid tags. The relatively short target segment allows the tag of each binder to be stably attached only to the capture probe. It can be set so that both binders cannot hybridize. When the two binder tags bind to the target protein and form an immune complex, They hybridize cooperatively to the capture probes sufficiently strongly to be stably captured. The approach also reduces nonspecific binders that are not part of the immune complex, which , reducing or even eliminating the need for release / recapture rounds.

[0378] Thus, in some embodiments, direct cooperative capture involves the binding of a first tag and a second tag. In the assay provided herein, the tags are cooperatively captured on a solid surface in step (1). The present invention provides a method for detecting an analyte in a sample, the method being used in a method for detecting an analyte in a sample (FIG. 6D). 1. An assay method for detecting a marker comprising the steps of: (1) mixing a first binder, a second binder, and the sample in a solution, the first and second binders bind to non-interfering epitopes on the analyte; and and forming a complex, wherein the immunocomplex is conjugated to the first binder. A first nucleic acid tag ("first tag") and a nucleic acid capture probe ("probe") coupled to the surface. a second nucleic acid tag ("second lobe") conjugated to the second binder; The probe is then hybridized to a solid surface in contact with the solution via hybridization between the probe and the tag. captured on the surface); (2) washing the solid surface to remove unbound molecules; and (3) detecting the immune complex. The assay method includes:

[0379] Cooperative capture can be repeated to further reduce non-specific binding. In an embodiment, the immune complex is characterized by cooperative hybridization between the first and second tags and the probe. The dissociation is then released from the first solid surface and attached to the second solid surface. and then cooperatively recaptured by the second solid surface, where the second solid surface is also coupled to the probe.

[0380] Cooperative capture can take a variety of forms. A contiguous fragment of a tag consists of a first fragment and an immediately adjacent second fragment, where the first tag and the When two tags are linked to form a linked nucleic acid, the junction region of the linked nucleic acid is The first fragment is complementary to the first tag or a contiguous fragment thereof. and the second fragment is complementary to the second tag or a fragment thereof. (FIG. 7A). In some embodiments, the first tag and the second tag are also attached to a second solid surface. In some embodiments, the contiguous fragments of the second probe are captured cooperatively. a fragment and a second fragment immediately adjacent thereto, wherein the first tag and the second tag are linked together. When forming a linked nucleic acid, the junction region of the linked nucleic acid is The first fragment is complementary to the first tag or a fragment thereof, so that the second fragment is complementary to the second tag or a fragment thereof. and the second fragment is complementary to the second tag or a fragment thereof. In some embodiments, the second probe is the same as the first probe. The immune complexes are then captured on a second solid surface through various mechanisms. The surface may be coupled with an antibody that binds to the first binder or the second binder. This can be done.

[0381] In some embodiments, the first fragment of the first probe is a first tag or a fragment thereof. a second fragment of the first probe that is complementary to the second tag or a fragment thereof; In some embodiments, the first tag and the second tag also In some embodiments, the second probe is cooperatively recaptured onto a second solid surface. The first fragment of the second probe is complementary to the first tag or a fragment thereof and is complementary to another fragment of the second probe. The second fragment is complementary to the second tag or a fragment thereof. The second probe is the same as the first probe. The regions are the unconjugated or "free" ends of the first and second tags, i.e., In some embodiments, the binder and the unconjugated end are included (Figure 6D). In this case, the complementary region does not include the unconjugated ends of the first and second tags (Figure 7B). In some embodiments, the complementary regions are conjugated to the first and second tags. In some embodiments, the second probe comprises a cleaved end (FIG. 7B). In some embodiments, the immune complex is transferred to the second antibody via various mechanisms. For example, the second surface is a solid surface of the first binder or the second binder. The antibody can be coupled to an antibody that binds to the antibody.

[0382] In some embodiments, direct cooperative capture is achieved by capturing a first solid surface onto a first probe. In the assay methods provided herein, the first probe is coupled to both the first probe and the second probe. (FIG. 7C). Provided herein are methods for detecting an analyte in a sample. 1. An assay method comprising the steps of: (1) mixing a first binder, a second binder, and the sample in a solution, the first and second binders bind to non-interfering epitopes on the analyte; and and forming a complex, wherein the immunocomplex is conjugated to the first binder. a first nucleic acid tag ("first tag") and a first nucleic acid capture probe coupled to the surface; ("first probe") and conjugated to the second binder. a gated second nucleic acid tag ("second tag") and a second nucleic acid coupled to the surface; The capture probe ("second probe") is brought into contact with the solution through hybridization between the capture probe and the second probe. be captured on a solid 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 associated with a second solid surface. and optionally recaptured, wherein the second solid surface also contains the first probe and the second probe. In some embodiments, the first solid surface is coupled to both the first a second solid surface coupled to the probe and an additional probe, and and an additional probe, wherein the first and second probes are Both hybridize with the first tag and the additional probe, and also hybridize with the second tag. In some embodiments, the second probe can be hybridized to the first In some embodiments, the immune complex is mobilized via various mechanisms. and captured on a second solid surface. For example, the second surface may be a first binder or a second binder. It can be coupled with an antibody that binds to the binder.

[0384] The complementary sequence between the nucleic acid tag and the nucleic acid capture probe allows for a specific hybridization between the tag and the probe. designed to promote the cleavage and subsequent dissociation of hybridized immune complexes. In one embodiment, the nucleic acid has an appropriate length and is sufficient for hybridization. It can provide sufficient strength and, upon release, can withstand low salt conditions without disrupting immune complexes. Sequences with few or no G / Cs that can dissociate under these conditions can be used. In some embodiments, sequences containing only poly T / A or poly "TA" / "AT" are used. As will be appreciated by those skilled in the art, appropriate hybridization strengths may be Any sequence having the following structure can be used.

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

[0386] In some embodiments, the assay methods provided herein are used More than 80%, more than 85%, more than 90%, more than 95%, or more than 100% of the complementary fragments of the universal probe and the nucleic acid capture probe are identical. In some embodiments, more than 98% of the amino acids are adenine ("A") and thymine ("T") pairs. , more than 80%, more than 85%, more than 90%, more than 95% of the complementary fragments of the universal probe and the first probe; More than 98% are A and T pairs. In some embodiments, the universal probe and the second capture More than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of the complementary fragments of the probe are A and T pairs. In some embodiments, more than 80% of the universal probe and first probe complementary fragments is an A and T pair, and more than 80% of the complementary fragments of the universal probe and the second probe are A and T. In some embodiments, the universal probe and the first probe complementary fragment are , A and T pairs, and the universal probe and second probe complementary fragments contain A and T pairs.

[0387] In some embodiments, the assay methods provided herein are used The complementary fragments of the nucleic acid tag and the nucleic acid capture probe are 10-30 base pairs, 10-25 base pairs, and 12-20 base pairs. In some embodiments, the amino acid sequence provided herein comprises 10 to 16 base pairs, or 10 to 16 base pairs. The complementary fragment of the nucleic acid tag and the nucleic acid capture probe used in the assay is 10 to 25 bases. In some embodiments, in the assay methods provided herein, The complementary fragment of the nucleic acid tag and the nucleic acid capture probe used consists of 12 to 20 base pairs. In some embodiments, the nucleic acid tags used in the assay methods provided herein The complementary fragment of the nucleic acid capture probe consists of 12 to 16 base pairs. and nucleic acid tags and nucleic acid capture probes used in the assay methods provided herein. In some embodiments, the complementary fragment of the first tag consists of 12 to 14 base pairs. The complementary fragment of one probe consists of 10 to 25 base pairs. The complementary fragment of the second tag and the second probe consists of 10 to 25 base pairs. In the above, the complementary fragment of the first tag and the first probe consists of 12 to 20 base pairs. In some embodiments, the complementary fragment of the second tag and the second probe is from 12 to 20 base pairs. In some embodiments, the complementary fragment of the first tag and the first probe is 12 to 18 carbon atoms. In some embodiments, the complementary sequence of the second tag and the second probe is 16 base pairs. The fragment consists of 12 to 16 base pairs.

[0388] (5.2.4 Detection) After sufficient removal of nonspecific binders that are not part of the immune complex by a capture / release mechanism Thus, the assay methods provided herein can detect immune complexes. includes detecting the immune complexes of step (6).

[0389] In some embodiments, the immune complexes are detected simultaneously as they are captured on a solid surface. The solid surface can be a second surface. At least one additional capture / release site If a solution 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, It is detected by.

[0390] In some embodiments, the assay methods provided herein involve the first step of detecting an immune complex. In some embodiments, the method provides for detecting a single binder. The assay method involves detecting a second binder of the immune complex. In an embodiment, the assay methods provided herein involve assaying a first binder of the immune complex. In some embodiments, detecting a first presentation group conjugated to the first presentation group. In the assay method provided herein, the first binder or the second binder of the immune complex is and detecting a second presentation group conjugated to one of the binders. In some embodiments, the immune complex comprises a detection antibody conjugated to a first binder. In some embodiments, the immune complex is detected via a second immunogenic marker. The detection is via a detectable marker conjugated to the binder.

[0391] In some embodiments, the detectable marker is detected by polymerase chain reaction (PCR). Protein analyte detection is performed using nucleic acid reporters, which are nucleic acids that can be amplified and detected by PCR (Figure 2). By converting the technology into a PCR probe, we have developed an immuno-PCR technology that utilizes the power of nucleic acid technology for protein detection. As shown in Figures 8B and 8D, The nucleic acid segment pre-conjugated to the antibody binder acts as a reporter of the immune complex. and PCR amplifies the reporter and produces a detectable signal. In some embodiments, the nucleic acid tag used for capture / release is In some embodiments, the antibody can be detected without the need for an additional detectable marker. In some embodiments, the first tag is used for detection by PCR. In this case, the second tag is used for detection by PCR.

[0392] Immune complexes can be detected by any method known in the art, for example The detection of the immune complex may be performed by using the first binder, the second binder, the first presentation group, or the second binder. This can be achieved by using an antibody that specifically binds to the presenting group of the or nearly colorless substrates or co-substrates to form colored complexes with highly colored products or chromogens. can be converted into a product that can be synthesized, e.g., horseradish peroxidase Can be labeled with enzymes, including alkaline phosphatase, or β-galactosidase 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 determined visually, spectrophotometrically, electrochemically, or by fluorescent It can be detected either optically or luminometrically and treated similarly. The detection system can also utilize radioactively labeled antibodies, In this case, the amount of immune complexes is determined by scintillation counting or gamma counting. Other detection systems that can be used include Staphylococcus aureus. Protein A from Staphylococcus aureus Cowan strain I, and protein B from group C Staphylococcus species (strain 26RP66) These include those based on the traditional use of protein G.

[0393] In some embodiments, the immune complexes are detected by immunofluorescence. In one embodiment, the immunoconjugate comprises a detectable marker conjugated to a 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 The colorimetric detectable marker can be a detection reagent or a chemiluminescent detection reagent. PNPP (p-nitrophenyl phosphate), ABTS (2,2'-azino-bis(3-ethylbenzothiazolinone) Examples include o-phenylenediamine (OPD), o-phenylenediamine (OPD), and o-phenylenediamine (OPD). Functional markers include QuantaBlu™ or QuantaRed™ (Thermo Scientific, Waltham Examples of luminescent detectable markers include luminol and luciferin. In some embodiments, the detectable marker includes a trigger (e.g., , H2O2) and tracers (e.g., isoluminol-conjugates). .

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

[0395] Methods and methods for performing immunoassays and biophysical protein-interaction assays Protocols and methods for immunoassays are well known in the art. 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, the nucleic acid tag is Conjugation to a binder for use in the assay method disclosed in the document Nucleic acid tags can be used to generate nucleic acid reporters that allow for highly sensitive detection. As noted above, provided herein are two-capture methods for detecting an analyte in a sample. 1. A binder assay method comprising: (1) mixing a first binder, a second binder, and the sample in a solution, the first and second binders bind to non-interfering epitopes on the analyte; and and forming a complex, wherein the immunocomplex is conjugated to the first binder. Hybridization between a first tag and a first probe coupled to the first surface. via a filtration onto a first solid surface in contact with the solution; (2) washing the first solid surface to remove unbound molecules; (3) The immune complex is subjected to hybridization between the first tag and the first probe. from the first solid surface via dissociation of (4) introducing the immunoconjugate to a second solid surface and conjugating the immunoconjugate to the second binder; The bond between the second tag attached to the second solid surface and the second probe coupled to the second solid surface is formed. recapture via conjugation; (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 for detecting an analyte in a sample. A method comprising: (1) mixing a first binder, a second binder, and the sample in a solution; The first and second binders bind to non-interfering epitopes on the analyte to form an immune complex. and wherein the immunocomplex is conjugated to the first binder. A first nucleic acid tag ("first tag") and a first nucleic acid capture molecule coupled to the first solid surface. The capture probe ("first probe") contacts the solution via hybridization between the capture probe and the first probe. (c) being captured on a first solid surface; (2) washing the first solid surface to remove unbound molecules; (3) The immune complex is subjected to hybridization between the first tag and the first probe. from the first solid surface via dissociation of (4) a second solid surface coupled with a second nucleic acid probe ("second probe"); and the immune complex is introduced into the first tag and the second probe by hybridization. recapturing the second solid surface via (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 also It is conjugated to a second nucleic acid tag (the "second tag").

[0399] In both the two-capture binder assay method and the one-capture binder assay method, the capture / Nonspecific primary and secondary binders that are not part of immune complexes by a release mechanism After sufficient removal of the reporter, a nucleic acid reporter is generated using the first tag and the second tag. Nucleic acid reporters can take a variety of forms. For example, the As shown, the first tag and the second tag are ligated to form a nucleic acid reporter. As will be appreciated by those skilled in the art, the present invention can be achieved by, for example, ligation, Polymerization extension, or cooperative hybridization, as disclosed herein or Any reporter generation method otherwise known in the art can be deployed at this step. do.

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

[0401] Proximity ligation assay (PLA) and proximity extension assay (PEA) are known in the art. (e.g., US6,511,809, US6,878,515, US7,306,904, US9,777,315, US10,17 4,366, WO9700446, Greenwood C, Biomol. Det. & Quan. 4(2015)10-16). Adjacency-based detection involves the binding of two nucleic acid conjugates to induce the formation of an amplifiable product. It differs from immuno-PCR in that it relies on the simultaneous recognition of target analytes by a cobinder. Thus, individual nucleic acid conjugate binders that are not part of the immune complex may generate reports. Therefore, background from single non-specifically bound binders is eliminated. In some embodiments, proximity ligation is used to generate nucleic acid reporters. (Figure 3A) where, upon formation of the immune complex, the first tag and the second tag are released. The fragments are sufficiently close to each other that they can be ligated to each other, and are composed of a fragment of the first tag and a fragment of the second tag. The resulting ligation product fragments are used as amplicons to generate a signal for detection. In some embodiments, proximity extension generates a nucleic acid reporter. (Figure 3B), where upon formation of the immune complex, the first tag and the second tag are bound together. The fragments are close enough to interact with each other to form a duplex, resulting in a minor fragment of the duplex. an enzyme for extending the 3' end of at least one nucleic acid tag to generate a signal for detection; Extension products can be generated that can be used as amplicons. In an embodiment, cooperative hybridization is performed to generate a nucleic acid reporter. In this case, the first tag and the second tag interact with each other upon formation of the immune complex. can be used as an amplicon to generate a signal for detection. In sufficient proximity to form a hybridization product.

[0402] The nucleic acid reporter is a nucleic acid tag that is used in step (1) of the assay method provided herein. As shown in Figure 4B(e), the reporter can be generated by combining a first tag with a The second tag can be attached via proximity ligation, proximity extension, or cooperative hybridization. Alternatively, the nucleotides shown in Figures 5A-5C can be generated by direct linkage. As shown, the nucleic acid reporter is a nucleic acid surrogate that can hybridize with the nucleic acid tag. For example, in some embodiments, the surrogate nucleic acid can be generated using wherein the first surrogate or a fragment thereof is complementary to the first tag or a fragment thereof. and the second alternative or fragment thereof is complementary to the second tag or fragment thereof. In embodiments, the reporter is a reporter that is a nucleotide sequence selected from the group consisting of proximity ligation, proximity extension, or cooperative hybridization. via cleavage to generate a first surrogate by linking the first surrogate to a second tag. In some embodiments, the reporter can be a nucleotide sequence selected from the group consisting of nucleotides ... , proximity extension, or cooperative hybridization to link a first tag to a second alternative. Alternatively, reporters can be generated by linking the reporters to the adjacent ligases (Figure 5B). The first surrogate is linked to the second surrogate via gating, proximity extension, or cooperative hybridization. This can be generated by linking two alternatives (Figure 5C). In the method, the first surrogate and / or the second surrogate are added after the immune complex is formed. In another embodiment, the first surrogate and the second surrogate are immunoconjugates. Before formation, they are pre-hybridized with a first tag and a second tag, respectively.

[0403] Thus, in some embodiments, the steps of the assay methods provided herein (6) by proximity ligation, proximity extension, or cooperative hybridization; linking the first tag and the second tag to form a nucleic acid reporter; and and detecting a nucleic acid reporter composed of a fragment of the first tag and a fragment of the second tag. In some embodiments, a surrogate nucleic acid is used, wherein the first tag or The fragment is complementary to the first surrogate or fragment thereof, and the second tag or fragment thereof is complementary to the first surrogate or fragment thereof. In some embodiments, the nucleotide sequence of the present invention is complementary to the nucleotide sequence of the present invention. Step (6) of the assay method is performed by proximity ligation, proximity extension, or cooperative hybridization. The nucleic acid receptor is formed by linking the first tag to the second surrogate via cleavage. generating a surrogate consisting of a fragment of the first tag and a fragment of the second surrogate; In some embodiments, detecting a nucleic acid reporter comprising the nucleic acid reporter provided herein. Step (6) of the assay method is performed by proximity ligation, proximity extension, or cooperative hybridization. The nucleic acid receptor is formed by linking the first surrogate to the second tag via cleavage. generating a surrogate consisting of a fragment of the first surrogate and a fragment of the second tag; In some embodiments, detecting a nucleic acid reporter comprising the nucleic acid reporter provided herein. Step (6) of the assay method is performed by proximity ligation, proximity extension, or cooperative hybridization. The nucleic acid residue is formed by linking the first surrogate to the second surrogate via cleavage. generating a surrogate comprising a fragment of the first surrogate and a fragment of the second surrogate; and detecting the nucleic acid reporter.

[0404] The assay methods exemplified in Figures 5A-5C and 8A-8B use proximity ligation. However, as noted above, proximity extension, cooperative hybridization, or other techniques known in the art may be used. Other methods 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. This can be done by PCR, quantitative PCR (qPCR), digital PCR (dPCR), or next-generation sequencing. In some embodiments, detection methods include, but are not limited to, next-generation sequencing (NGS). In some embodiments, the detection is a qualitative detection. In some embodiments, the detection is a 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 this case, the nucleic acid reporter is detected by NGS.

[0406] In addition to these common techniques, other nucleic acid amplification / detection methods can also be used, These include rolling cycle amplification (RCA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), and Target amplification includes, but is not limited to, PCR amplification and recombinase polymerase amplification (RPA). Further techniques that allow for highly sensitive nucleic acid detection without the need for broadband are disclosed in the assays disclosed herein. These may also be employed for the detection of nucleic acid reporters in methods. QuantiGene assay from Fisher Scientific (ThermoFisher (2019)), SIMOA from Quanterix assay (Rissin DM (2010)), and SMCxPRO™ from MilliporeSigma (MilliporeSigma (20 19)). Thus, in some embodiments, Nucleic acid reporters can be synthesized using a variety of methods including rolling cycle amplification (RCA), strand displacement amplification (SDA), and loop-mediated isothermal amplification (LAMP). Detected by loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), or QuantiGene assays It is served.

[0407] (5.2.5 Multiplexing) The reporters generated in the assay methods disclosed herein are nucleic acid molecules. The unique sequence can be deciphered by DNA sequencing or other methods. It can be incorporated as a separate barcode (ID). ID segments containing N base nucleotides The maximum number of comments is 4. N One approach is to generate a unique identification code for each As shown in Figures 9A-9B, the ID can be attached to a tag on one binder (Figure 9A) or both binders. In some embodiments, the nucleic acid label is directly incorporated into the tag of the nucleic acid label (Figure 9B). In some embodiments, the reporter contains an ID in the first tag. In some embodiments, the nucleic acid reporter comprises an ID in the first tag. The tag contains a first ID and the second tag contains a second ID.

[0408] FIG. 10 shows another single-stranded nucleic acid molecule ( This paper presents an indirect ID barcoding approach based on nucleic acid surrogates. In some embodiments, an alternative nucleic acid is used, in which case the first tag or a fragment thereof is a first tag or fragment thereof complementary to a second surrogate or In Figure 10A, only the first binder is bound by the first alternative. The nucleic acid reporter ligates the first surrogate to a second tag. In FIG. 10B, the first binder and the second binder are Both the nucleotides are indirectly ligated. The ligation is exemplified in Figures 9A-9B and 10A-10B. Although the reporter is shown to be a nucleotide sequence that is amplified by the proximity extension, cooperative hybridization, and The ID may also be generated by other methods known in the art. Annealing of the nucleic acid surrogate with its associated binder tag is performed during reagent preparation prior to the assay. This can be done in a process, or it can be done as part of an assay. .

[0409] In some embodiments, provided herein is a nucleic acid reporter comprising a first Assay methods containing an identifier in a tag, a first surrogate, a second tag, or a second surrogate. In some embodiments, the nucleic acid reporter comprises a fragment of a first tag and a second surrogate. and contains an ID in the first tag or second alternative. In an embodiment, the nucleic acid reporter comprises a first identifier in a first tag and a second identifier in a second alternative. In some embodiments, the nucleic acid reporter comprises a fragment of the first surrogate and a fragment of the second tag, and contains an ID in the first alternative or second tag. In some embodiments, the nucleic acid reporter comprises a first ID and a second ID in a first alternative. In some embodiments, the nucleic acid reporter contains a second ID in the first alternative. It is composed of a fragment of a substitute and a fragment of a second substitute, and the first substitute or the second substitute In some embodiments, the nucleic acid reporter contains an ID in the first alternative. Contains a first ID and a second ID in the second alternative.

[0410] One application of the above-described ID methods is the parallel detection and measurement of multiple different analytes in the same sample. In some embodiments, the nucleic acid reporters disclosed herein are , contains an analyte-specific ID ("target ID"). Each analyte is assigned a unique ID Thus, provided herein is a method for simultaneously determining the unique target ID associated with each analyte. simultaneously detecting at least two analytes in the sample by simultaneously detecting In some embodiments, the assay methods provided herein are The method detects at least 100 unique analytes in a sample by simultaneously detecting the unique target ID associated with each analyte. At least 3, at least 4, at least 5, at least 6, at least 7, at least 8 one, at least nine, 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 are detected simultaneously.

[0411] In some embodiments, the analyte is a protein. In this case, the analyte comprises at least one protein and at least one nucleic acid. The nucleic acid is a DNA fragment. The assay methods provided herein can be used to assay proteins, DNA, The fact that it can be used for the analysis of RNA and RNA makes the method suitable for multi-omics analysis. This provides an ideal platform for analysis.

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

[0413] Incorporation of ID into nucleic acid reporters in the assay methods provided herein allows for This can also help improve the specificity of the first Associate each of the binder and the second binder with a unique ID, and the IDs of both binders are Only signals generated from reporters that contain the reporter are counted as true signals. Using the scheme of False positive signals can be reduced or eliminated. a first binder and a second binder, respectively, each associated with a first label; US201301229144 - Assay method for detecting an analyte in a sample by simultaneous detection of a target ID and a second target ID - Google Patents be.

[0414] Incorporation of ID into nucleic acid reporters in the assay methods provided herein allows for the identification of molecules Use to detect interactions between proteins, e.g., protein-protein interactions As shown in Figure 13, two binders designed for various molecules are Simultaneous detection and quantification of the reporter containing the ID of these two related proteins under assay conditions In some embodiments, the methods described herein will demonstrate molecular interactions and affinities. The assay methods provided detect protein-protein interactions. Provided herein is an assay method for detecting an analyte in a sample, wherein and the analyte is a binding pair of two different proteins; where the first binder is One binder binds to one protein, and the second binder binds to the other protein in the binding pair. and wherein the binding pair is detected by simultaneous detection of the first target ID and the second target ID. do.

[0415] In addition to the analyte-specific "target ID," the The nucleic acid reporters used may also include a sample-specific "sample ID." As described above, when the assay methods provided herein are performed on a particular sample, A sample ID is introduced during the assay at or before the reporter generation step to identify the sample. By incorporating such a sample ID into the reporter, it is possible to identify the sequence of many samples. Several reporters can be pooled together and read in parallel by NGS. In some embodiments, the sample ID is carried on the nucleic acid independently of the binder tag, as shown in FIG. can be incorporated into the reporter in the ligation step shown in Figure 1. In some embodiments, the nucleic acid reporter formed in each sample is a sample ID. wherein the sample ID is between a first tag or surrogate and a second tag or surrogate. is inserted into

[0416] In some embodiments, the nucleic acid reporter comprises a fragment of a first tag and a fragment of a second tag. The tag is made up of a first tag and a second tag and contains a sample ID inserted between the first tag and the second tag. In some embodiments, the nucleic acid reporter comprises a fragment of a first tag and a fragment of a second surrogate. a sample ID inserted between the first tag and the second alternative; In some embodiments, the nucleic acid reporter comprises a fragment of a first surrogate and a fragment of a second tag. fragment and containing a sample ID inserted between the first alternative and the second tag. In some embodiments, the nucleic acid reporter comprises a fragment of a first surrogate and a fragment of a second surrogate. a sample ID consisting of a fragment of a substance and inserted between the first substitute and the second substitute; Contains.

[0417] Alternatively, the sample ID may be hybridized to the tag during the assay step shown in Figure 14B. Thus, in some embodiments, The nucleic acid reporter formed in each sample is a sample in the first surrogate or the second surrogate. In some embodiments, the nucleic acid reporter comprises a fragment of the first tag and a fragment of the second tag. It is composed of fragments of two surrogates and contains the sample ID in the second surrogate. In an embodiment, the nucleic acid reporter is comprised of a first surrogate fragment and a second tag fragment. and contains a sample ID in the first alternative. The target is composed of a fragment of a first substitute and a fragment of a second substitute, and is In the second alternative, include the sample ID.

[0418] Alternatively, the sample ID can be provided by a nucleic acid tag or a substitute thereof during the assay step shown in FIG. 14C. The nucleic acid can be incorporated by ligation into the nucleic acid. In the method, the nucleic acid reporter formed in each sample is a first tag or a second tag. , or its respective alternatives, containing the sample ID ligated to In embodiments, the nucleic acid reporter is comprised of a fragment of a first tag and a fragment of a second tag; and containing a sample ID ligated to the first tag or the second tag. In this embodiment, the nucleic acid reporter comprises a first tag fragment and a second surrogate fragment. and containing a sample ID ligated to the first tag or the second alternative. In some embodiments, the nucleic acid reporter comprises a fragment of a first surrogate and a fragment of a second tag. a sample ID composed of fragments and ligated to the first surrogate or the second tag; In some embodiments, the nucleic acid reporter comprises a fragment of a first surrogate and a fragment of a second surrogate. A fragment of a second surrogate is composed of fragments of the first surrogate or the second surrogate. Contains the sample ID.

[0419] Accordingly, also provided herein are nucleic acid reporters formed in each sample. Incorporating a unique sample ID into the sample and a unique nucleic acid reporter associated with each sample Simultaneous detection of analytes in at least two samples by simultaneously detecting the sample IDs of the samples. In some embodiments, the assay method provided herein comprises: The assay method allows for simultaneous detection of the unique sample ID associated with each sample, thereby minimizing the number of samples. at least three, at least four, at least five, at least six, at least seven, at least 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 analytes in the sample Detect simultaneously.

[0420] In some embodiments, the assay methods provided herein are A nucleic acid reporter can include both a target ID and a sample ID. wherein the nucleic acid reporter is (a) in a first tag or a first surrogate, or in a second tag or (b)(1) the target ID in the first tag or its surrogate and the second tag; or (2) inserted between the first or second substitutes; or (3) the first tag or its substitute or the second tag or its substitute Contains the sample ID that is being ligated to the substitute.

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

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

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

[0424] In some embodiments, the nucleic acid reporter comprises a fragment of a first surrogate and a fragment of a second surrogate. and contains a target ID and a sample ID. or the second surrogate may contain a target ID. The nucleic acid reporter may be a nucleic acid reporter in the first surrogate. The nucleic acid report may contain a first target ID in one alternative and a second target ID in a second alternative. The controller may contain a sample ID inserted between the first and second alternatives. The nucleic acid reporter can also contain a sample ID in the first alternative or the second alternative. .

[0425] Thus, provided herein is a unique sample ID and unique By simultaneously detecting the unique target ID and the unique sample ID associated with each sample, at least and also assay methods involving simultaneously detecting at least two analytes in two samples. do.

[0426] In some embodiments, the assay methods provided herein comprise: By simultaneously detecting the unique sample ID and the unique target ID in the corresponding nucleic acid reporter, at least three, at least four, at least five, at least six, at least seven, at least 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 The two analytes are detected simultaneously.

[0427] In some embodiments, the assay methods provided herein comprise: By simultaneously detecting the unique sample ID and the unique target ID in the corresponding nucleic acid reporter, at least three, at least four, at least five, at least six, at least seven, at least 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 The three analytes are detected simultaneously.

[0428] In some embodiments, the assay methods provided herein comprise: By simultaneously detecting the unique sample ID and the unique target ID in the corresponding nucleic acid reporter, at least three, at least four, at least five, at least six, at least seven, at least 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 Five analytes are detected simultaneously.

[0429] In some embodiments, the assay methods provided herein comprise: By simultaneously detecting the unique sample ID and the unique target ID in the corresponding nucleic acid reporter, at least three, at least four, at least five, at least six, at least seven, at least 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 10 analytes are detected simultaneously.

[0430] In some embodiments, the assay methods provided herein comprise: By simultaneously detecting the unique sample ID and the unique target ID in the corresponding nucleic acid reporter, at least three, at least four, at least five, at least six, at least seven, at least 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 20 analytes are detected simultaneously.

[0431] In some embodiments, the assay methods provided herein comprise: By simultaneously detecting the unique sample ID and the unique target ID in the corresponding nucleic acid reporter, at least three, at least four, at least five, at least six, at least seven, at least 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 50 analytes are detected simultaneously.

[0432] In some embodiments, the assay methods provided herein comprise: By simultaneously detecting the unique sample ID and the unique target ID in the corresponding nucleic acid reporter, at least three, at least four, at least five, at least six, at least seven, at least 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 80 analytes are detected simultaneously.

[0433] In some embodiments, the assay methods provided herein comprise: By simultaneously detecting the unique sample ID and the unique target ID in the corresponding nucleic acid reporter, at least three, at least four, at least five, at least six, at least seven, at least 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 100 analytes are detected simultaneously.

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

[0435] The nucleic acid reporter in the multiplexed assay method disclosed herein is Detect by multiplexed qPCR, multiplexed digital PCR, or NGS For example, in some embodiments, the multiplexing methods disclosed herein can be used. The nucleic acid reporter in the sequencing assay method can be detected by NGS. NGS for detecting nucleic acid reporters generated by the assay methods disclosed herein The use of NGS has at least the following advantages: First, NGS detects the sequence of nucleic acid molecules in a sample. The target and sample ID tags can be read as described herein. By incorporating these technologies into a single platform, NGS enables multiplexed detection on a very large scale. For example, NGS can be performed on a pool of 100 samples each containing 10 targets (i.e. , 1000-plex) can be read in one run. This means that Second, NGS allows counting and summing the number of molecules with the same sequence. In combination, this can provide digital quantification at single molecule resolution. Because it is predesigned and short, NGS as a reporter detection method is not suitable for de novo Compared to sequencing, it can be completed much faster and at a lower cost. Parity check, Hamming code (e.g., Bystrykh, PLoS ONE 7(5): e36852(2 012)), and Levenshtein coding (see, e.g., Buschmann, BMC Bioinformatics. 2013; 14: 272(2013)) and uses a wide variety of error correction algorithms from communication theory. , which can be applied herein to reduce false positive counts, thereby improving the accuracy of NGS-based Quantitation can be achieved with high accuracy without repeated sequencing.

[0436] Since NGS is a single molecule detection and counting method, the sequencing device It imposes an upper limit on the total number of molecules that can be sequenced. The Illumina MiSeq system For example, 25 million reads per run are possible, which allows The total number of molecules sequenced is limited to 25 million. This limitation is due to the low target abundance. If present at or near the limit of detection ("LOD"), it is not limiting for most applications. However, in multiplexed studies, some targets are orders of magnitude more potent than others. Highly expressed and unsequenced genes that do not provide useful clinical / biological information. It is known that multi-analyte sensitivity consumes bandwidth. The signals generated from these large numbers of analytes can be analyzed while maintaining a plexed assay format. It is necessary to intentionally lower the

[0437] The assay methods provided herein further address this need and provide related advantages. In some embodiments, provided herein are methods for detecting a target molecule at a high concentration. The number of reporter molecules generated from the precipitate is reduced by a precise and known percentage, resulting in a limiting and an assay method that can efficiently allocate a given detection bandwidth to different target analytes. The assay methods disclosed herein involve the addition of an acceptor group (e.g., a nucleotide sequence) to a reporter molecule before the reporter is generated. For example, nucleic acid capture probes are used to capture the immune complexes onto a solid surface, from which they can be released. By selectively capturing a small portion of the immune complexes that are generated on the surface, a very large amount of This provides a unique opportunity to reduce the analyte signal.

[0438] For example, in some embodiments, a binder can be attached to its presentation group (e.g., a nucleic acid tag). For example, the presenting group and the conjugate can be combined in known proportions. When the jugated binder was mixed with the same binder without the presenting group at a concentration of 1% , only 1% of the immune complexes could be captured on the surface, and the reporter-to-target analyte ratio was 1%. Non-functional presenting groups, i.e., presenting groups that do not bind to an acceptor group, can also be used. For example, no more than 0.1% of the first binders contain functional first presenting groups (e.g., first tag), and the remaining 99.9% of the first binder is conjugated to a first acceptor group (e.g., a first presentation group (e.g., a first tag) that cannot be captured by a first probe When conjugated to the analyte, the reporter to immune complex ratio is 1:1000. effectively reducing the signal generated by 1000-fold.

[0439] Incorporating a known portion of a non-functional acceptor group (e.g., a nucleic acid capture probe), such a portion Another approach for indirect capture can be used. In the universal capture method, for example, the universal capture probe does not have a segment complementary to the universal capture probe (Figure 6 B) or not biotinylated (Figure 6C), allowing the inclusion of a specific proportion of first capture probes. As a result, the same proportion of immune complexes cannot be captured on the surface and therefore For example, if the acceptor group is not attached to a solid surface, it is not possible to generate a nucleic acid reporter for detection. It contains only 0.1% functional molecules that can be coupled (the remaining 99.9% are non-functional). In the case of a reporter-to-immunocomplex (a functional mimetic molecule), the reporter-to-immunocomplex ratio is also 1:1000, and This effectively reduces the signal generated by 1000 times.

[0440] Therefore, provided herein is a method for preparing a polymerizable composition comprising adding a non-functional binder to a solution in step (1). by reducing the amount of analyte detected by the assay. wherein the non-functional binder competes with the first binder for binding to the analyte. , unconjugated or conjugated to a presenting group that does not bind to the first acceptor group In some embodiments, the non-functional In some embodiments, the non-functional binder is not conjugated. The initiator is conjugated to a presenting group that does not bind to the first acceptor group. In this embodiment, the non-functional binder is a first probe coupled to a first solid surface. The nucleic acid tag is conjugated to a nucleic acid tag that cannot hybridize to the antibody.

[0441] Therefore, provided herein is a method for preparing a soluble polymeric polymer comprising adding a non-functional receiver to a solution in step (1). by reducing the amount of analyte detected by the assay. wherein the non-functional receiver competes with the first acceptor for binding to the first presentation group. , is also an assay method in which the first solid surface cannot be coupled to the second solid surface.

[0442] In some embodiments, provided herein is a method for producing a non-functional vine in step (1). Adding a dye to the solution proportionally reduces the amount of analyte detected by the assay. wherein the non-functional binder inhibits binding to the analyte from a first binder. Immune complexes that compete with either the primary or secondary binder but cannot be detected. In some embodiments, the methods provided herein are also assay methods for forming a The assay method involves the use of a compound conjugated to either the first binder or the second binder. The immune complexes are detected by detecting the attached detectable marker, and non-functional viable cells are detected. The indicator is not conjugated to a detectable marker or does not produce a reporter for detection. Some are conjugated to defective detectable markers that cannot be produced. In an embodiment, the immune complexes are detected via a nucleic acid reporter, and non-functional binders are detected. The nucleotide sequence is conjugated to a nucleic acid tag that lacks the appropriate segment to generate a nucleic acid reporter. Those skilled in the art will appreciate that the amount of analyte in a sample can be significantly reduced by the amount of analyte produced. In order to proportionally reduce the signal generated by the ion beam, various variations of the methods disclosed herein may be used. approach, allowing for the simultaneous detection of multiple analytes that may be present at concentrations differing by many orders of magnitude. You will understand that it is possible.

[0443] In addition to NGS, many other single molecule detection techniques, such as digital PCR and SIMOA, can be performed in a single run. There is a limit to the total number of target molecules that can be read by the system (instrument bandwidth). The above method of precisely controlling the antibody / immunoconjugate ratio maximizes the efficient use of system resources. Therefore, it may be applicable to all of these techniques.

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

[0445] As described in Section 5.1, target labels can be associated with specific labels. Identifiers can be provided to facilitate detection and identification of target molecules. Methods provided herein, including those described in Section 5.2.6 and the preceding paragraph. In some embodiments, each target label can include one or more target IDs. In one specific embodiment, the first target label comprises a first target ID. In one embodiment, the second target label comprises a second target ID. One target label includes a first target ID, and the second target label includes a second target ID.

[0446] As further described in Section 5.2.4 above, including those in this section (Section 5.2.6) In some embodiments of the methods provided herein, the reporter is a first target can be generated based on the proximity between the label and a second target label. In an embodiment, the reporter comprises a first target ID. In yet another embodiment, the reporter comprises a first target ID. In a further embodiment, the reporter comprises a first target ID and 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 an 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 for detecting an analyte in a sample. A method comprising: (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the groups; and (iii) the first binding moiety binds to a first identification barcode (“ID”) that is analyte-specific (“Target ID”) ") and the second binding moiety comprises a second target ID. further comprising a second target label); (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 (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 Section 5.2.4.1 and Figures 3A-3C, among others, the present invention provides In various embodiments of the method, the reporter is a binding site between both the binder and the analyte. This can be generated based on the properties of the immune complex that correlate with the specificity of binding. In one embodiment of the methods provided herein, including those in Section 2.6, the reporter is: The target is generated based on the proximity between the first target label and the second target label. In this case, a reporter is generated based on the proximity between a first target ID and a second target ID. In yet another embodiment, the reporter is a molecule between the first binder and the second binder. Generated based on proximity.

[0449] In one aspect, provided herein is an assay for detecting an analyte in a sample. A method comprising: (1) a first binding portion including a first binder and a second binding portion including a second binder; mixing 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 binds to a first identification barcode (“ID”) that is analyte-specific (“Target ID”) ") and the second binding moiety comprises a second target ID. further comprising a second target label); (2) generating a reporter from the immune complex, wherein the reporter is a first target based on the proximity between the reporter and a second target label, and wherein the reporter comprises: (i) a first target ID, (ii) a second target ID, or (iii) both a first target ID and a 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, the present specification, including that of this section (Section 5.2.6), In some embodiments of the methods provided herein, the reporter is a non-nucleic acid reporter. In other embodiments, the reporter is a nucleic acid reporter. In a specific embodiment of the method provided herein, (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the groups; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprises a further comprising a second target label; (2) washing the first solid surface to remove unbound molecules; (3) extracting nucleic acid fragments from the immune complex based on the proximity between the first target label and the second target label. generating a reporter; and (4) detecting the reporter, thereby detecting the analyte. Contains: In another specific embodiment, the methods provided herein comprise: (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the groups; and (iii) the first binding moiety binds to a first identification barcode (“ID”) that is analyte-specific (“Target ID”) ") and the second binding moiety comprises a second target ID. further comprising a second target label); (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 is (ii) a first target ID, or (iii) a second target ID, or both a first target ID and a second target ID. );and (4) detecting the reporter, thereby detecting the analyte. Contains:

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

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

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

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

[0457] Additionally, immune complexes can be released from the first surface, as described above in Section 5.2.1.1. The antibody can be allowed to react with the antibody and recaptured on a second surface to further increase the signal to noise ratio. 21A-21F show exemplary schematics of such a two-capture assay method. Thus, in one embodiment of the methods provided herein, the second binding moiety is In another embodiment, the method further comprises: Step 2(b): (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; The immune complex is recaptured on the second solid surface via binding between the linked second acceptor groups. Thus, in some embodiments, the methods provided herein further comprise capturing The way to do this is (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprising a second target label); (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor group. ; (3) detecting a receptor from the immune complex based on the proximity between the first target label and the second target label; generating a neutron; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0458] In other embodiments, the methods provided herein comprise: (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety contains a first identification barcode (“ID”) that is analyte-specific (“target”) and the second binding moiety further comprises a first target label comprising a second target ID. further comprising a second targeting label; (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor group. ; (3) generating a reporter from the immune complex, wherein the reporter comprises: (i) the first (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 may include a step 2(c) between step 2(b) and step (3):(2c) a second Further comprising washing the solid surface to remove unbound molecules. In an embodiment, the methods provided herein comprise: (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprising a second target label); (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor group. ; (2c) washing the second solid surface to remove unbound molecules; (3) detecting a receptor from the immune complex based on the proximity between the first target label and the second target label; generating a neutron; and (4) detecting the reporter, thereby detecting the analyte. Contains:

[0460] In other embodiments, the methods provided herein comprise: (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety contains a first identification barcode (“ID”) that is analyte-specific (“target”) and the second binding moiety further comprises a first target label comprising a second target ID. further comprising a second targeting label; (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor group. ; (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 (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] Further, the methods provided herein may include step 2(d): a step of reacting a second presenting group with a second accepting group. The method further includes releasing the immune complex from the second solid surface by disrupting the bond. Thus, in some embodiments, the methods provided herein include: (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprising a second target label); (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor group. ; (2c) washing the second solid surface to remove unbound molecules; (2d) disrupting the bond between the second presenting group and the second accepting group, thereby releasing from said second solid surface; (3) detecting a receptor from the immune complex based on the proximity between the first target label and the second target label; generating a neutron; and (4) detecting the reporter, thereby detecting the analyte. Contains:

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

[0464] In yet another embodiment, the methods provided herein comprise: (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety contains a first identification barcode (“ID”) that is analyte-specific (“target”) and the second binding moiety further comprises a first target label comprising a second target ID. further comprising a second targeting label; (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor group. ; (2c) washing the second solid surface to remove unbound molecules; (3) disrupting the bond between the second presenting group and the second accepting group, thereby releasing the immunocomplex from the second solid surface and generating a reporter from the immunocomplex (this wherein the reporter is (i) the first target ID, (ii) the second target ID, or (iii) the first target ID. 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) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to the analyte to form an immune complex; (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety further comprises a first target label, and the second binding moiety further comprising a second target label); (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor group. ; (2c) washing the second solid surface to remove unbound molecules; (3) isolating a receptor from the immune complex based on the proximity between the first target label and the second target label. To generate porters; (4) disrupting the bond between the second presenting group and the second accepting group, thereby releasing it from the second solid surface; and (5) detecting the reporter, thereby detecting the analyte. Contains:

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

[0467] In one aspect, provided herein is an assay for detecting an analyte in a sample. A method comprising: (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to non-interfering epitopes on the analyte; and Forming a complex, (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety contains a first identification barcode (“ID”) that is analyte-specific (“target”) and the second binding moiety further comprises a first target label comprising a second target ID. further comprising a second targeting label; (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] Detecting immune complexes and nucleic acids as described above in Sections 5.2.1.1 and 5.2.1.2. The reporter is generated either on the solid surface or after release from the solid surface. 20A-20D show the structure of a nucleic acid reporter (shown as the first surface in FIGS. 20B-20C). 20E provides an exemplary schematic of the generation of such an exemplary capture and release provides confirmatory data showing from assays in which the analyte is a nucleic acid The target is detected by a signal determined by the target ID of the target label generated in the transporter. Thus, in one embodiment, the method provided herein comprises steps (2) and (3). Step (2a): By breaking the bond between the first presenting group and the first accepting group, the immunoconjugate Further comprising releasing the body from the first solid surface. , the methods provided herein comprise: (1) a first binder and a first binding moiety including a first presenting group and a second binder; mixing a second binding moiety and the sample in solution, wherein: (i) the first and second binders bind to non-interfering epitopes on the analyte; and Forming a complex, (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety contains a first identification barcode (“ID”) that is analyte-specific (“target”) and the second binding moiety further comprises a first target label comprising a second target ID. further comprising a second targeting label; (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (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] Additionally, immune complexes can be released from the first surface, as described above in Section 5.2.1.1. The antibody can be allowed to react with the antibody and recaptured on a second surface to further increase the signal to noise ratio. 21A-21F show exemplary schematics of such a two-capture assay method. Thus, in one embodiment of the methods provided herein, the second binding moiety is In another embodiment, the method further comprises: Step 2(b) and Step 2(c): (2b) introducing a second solid surface and connecting a second presenting group to the second solid surface; The immune complex is attached to the second solid via binding between the second acceptor groups coupled to the surface. (2c) washing the second solid surface to remove unbound molecules. Thus, in some embodiments, the methods provided herein further comprise: teeth, (1) a first binder and a first binding moiety including a first presenting group and a second binder and mixing the sample in solution with a second binding moiety comprising two presentation groups, wherein: (i) the first and second binders bind to non-interfering epitopes on the analyte; and Forming a complex, (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety contains a first identification barcode (“ID”) that is analyte-specific (“target”) and the second binding moiety further comprises a first target label comprising a second target ID. further comprising a second targeting label; (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor group. ; (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 is detected, and the nucleic acid reporter is detected either on the solid surface or after being released from the solid surface. 21G-21I show the capture and release of the first and second solid surfaces. 24A-24B and 25A-25C provide exemplary schematic diagrams of the generation of nucleic acid reporters after provides corroborative data showing such exemplary capture and release assays, In (a), the analyte is determined by the target ID in the target label generated in the nucleic acid reporter. Thus, in one embodiment, the present invention provides The method further comprises step (2d): breaking the bond between the second presenting group and the second accepting group. and further comprising releasing the immune complexes from the second solid surface. In the manner provided herein, the method comprises: (1) a first binder and a first binding moiety including a first presenting group and a second binder and mixing the sample in solution with a second binding moiety comprising two presentation groups, wherein: (i) the first and second binders bind to non-interfering epitopes on the analyte; and Forming a complex, (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety contains a first identification barcode (“ID”) that is analyte-specific (“target”) and the second binding moiety further comprises a first target label comprising a second target ID. further comprising a second targeting label; (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor group. ; (2c) washing the second solid surface to remove unbound molecules; (2d) disrupting the bond between the second presenting group and the second accepting group, thereby releasing from said second solid surface; (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] In addition to the analyte-specific "target ID," as further described in Section 5.2.5 above, The nucleic acid reporters generated in the assay methods provided herein are sample-specific "test" 22 and 23A-23B show a nucleic acid reporter having a sample ID. 24A-24B and 25A-25C provide exemplary schematic diagrams for creating such multi- provides confirmatory data showing a plexed assay in which the analyte is a sequence determined by the target ID and sample ID in the target label generated in the nucleic acid reporter. The samples are detected by the signal, allowing the samples to be analyzed by sample ID and separated by target ID. The precipitate is analyzed. Thus, in one embodiment, the method provided herein comprises the steps of: (2e): A sample label containing a sample-specific ID ("sample ID") is added to (i) a first target label and (ii) a second target label. or (iii) to both the first target label and the second target label. In one specific embodiment, the methods provided herein further comprise: (1) a first binder and a first binding moiety including a first presenting group and a second binder and mixing the sample in solution with a second binding moiety comprising two presentation groups, wherein: (i) the first and second binders bind to non-interfering epitopes on the analyte; and Forming a complex, (ii) the immune complex is coupled to the first presenting group and a first receptor coupled to a first solid surface; is captured on the first solid surface in contact with the solution via a bond between the soluble groups; and (iii) the first binding moiety contains a first identification barcode (“ID”) that is analyte-specific (“target”) and the second binding moiety further comprises a first target label comprising a second target ID. further comprising a second targeting label; (2) washing the first solid surface to remove unbound molecules; (2a) disrupting the bond between the first presenting group and the first accepting group, thereby releasing from said first solid surface; (2b) introducing a second solid surface and coupling a second presenting group to the second solid surface; and recapturing the immune complex to the second solid surface via binding between the second acceptor group. ; (2c) washing the second solid surface to remove unbound molecules; (2d) disrupting the bond between the second presenting group and the second accepting group, thereby releasing from said second solid surface; (2e) a sample label comprising an ID ("sample ID") that is sample-specific to (i) the first target label; (ii) (iii) to the second target label, or (iv) to both the first target label and the second target label. To (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 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 one embodiment, the first binding moiety comprises five or more target labels. In another embodiment, the first binding moiety comprises one target label. 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 some embodiments, 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. The target label between the first binding moiety and the second binding moiety is the same. In some embodiments, the target labels in the first binding moieties are different. In some further embodiments, the target labels in the binding moiety are identical. In other embodiments, the target labels in the second binding moieties are the same. In some embodiments, the first binding moiety is any target provided in this paragraph. and the second binding moiety has any of the target labels provided in this paragraph in any combination. In one specific embodiment, the first binding moiety has a first a first binding moiety comprising a target label and a second binding moiety comprising a second target label, wherein the first target label and the second target label are different. In another specific embodiment, the first binding moiety is and the second binding moiety comprises a second target label, wherein the first target label The target marker and the second target marker are identical.

[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 another embodiment, the second binding moiety lacks a target label. In some embodiments, the first binding moiety does not comprise a target label. The second binding moiety does not comprise a target label. In another embodiment, the first binding moiety does not contain a label and the second binding moiety contains one target label. One binding moiety contains one target label and the second binding moiety does not contain a target label. In this embodiment, the first binding moiety does not include a target label and the second binding moiety includes two, three, or more target labels. In another embodiment, the first binding The moiety comprises two, three, four, five or more target labels, and the second binding moiety comprises In one embodiment, the first binding moiety lacks a target label and the second The binding moiety comprises one target label. In another embodiment, the first binding moiety comprises one target In another embodiment, the first binding moiety comprises a target label and the second binding moiety lacks a target label. The binding moiety lacks a target label and the second binding moiety has two, three, four, five, or more In another embodiment, the first binding moiety comprises two, three, four, five, or more target labels, and the second binding moiety lacks the target label.

[0474] Similarly, one of the two binding moieties may not contain a presenting group. In one embodiment of the methods provided herein, including those in which the first binding moiety is In another embodiment, the second binding moiety lacks a presenting group. In another embodiment, the first binding moiety does not include a presenting group. In one embodiment, the first binding moiety does not include a presenting group. In another embodiment, the first binding moiety comprises one presenting group. In one embodiment, the first binding moiety comprises one presenting group and the second binding moiety does not comprise a presenting group. The binding moiety lacks a presenting group and the second binding moiety contains one presenting group. wherein the first binding moiety comprises one presenting group and the second binding moiety lacks a presenting group.

[0475] Additionally, the methods provided herein, including those in this section (Section 5.2.6) and the preceding paragraph, In some embodiments, each target label can include one or more target IDs. In one embodiment, the first target label comprises a first target ID. The second target label comprises a second target ID. In some embodiments, the first target label In some embodiments, the target ID between the first target label and the second target label is different. In one specific embodiment, the target IDs between the first and second target labels are identical. The identification includes a first target ID and the second target identification includes a second target ID, wherein the first In another specific embodiment, the first target label is: a first target ID and a second target label comprising 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, including those in this section (Section 5.2.6), In some embodiments of the methods provided herein, each nucleic acid reporter is , can include one or more target IDs and / or sample IDs. In one embodiment, in each sample The nucleic acid reporter formed contains a sample ID. In a further embodiment, each of the nucleic acid reporters formed by The nucleic acid reporter formed in the sample contains the first target ID. In this case, the nucleic acid reporter formed in each sample contains a second target ID. In this embodiment, the nucleic acid reporter formed in each sample is associated with a sample ID and a first target. In one embodiment, the nucleic acid reporter formed in each sample contains a target ID. In another embodiment, the nuclei formed in each sample contain a sample ID and a second target ID. The acid reporter contains a first target ID and a second target ID. 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, each sample The nucleic acid reporter formed in contains a first target ID, a second target ID, and a sample ...

Claims

1. 1. An assay method for detecting an analyte in a sample, comprising: Step (1) combining a first binder and a first binding moiety including a first presenting group with a second binder. mixing the sample in solution with a second binding moiety comprising: (i) the first and second binders bind to the analyte to form an immune complex; (ii) The immune complex is attached to a first solid surface in contact with the solution, the first presenting group and the first solid captured via a bond between a first receptor group coupled to a body surface; and (iii) the first binding moiety comprises a first identification barcode (“ID”) (“first and the second binding moiety further comprises a first target label comprising a second target ID ("Target ID"). further comprising a second target label comprising D); step (2) washing the first solid surface to remove unbound molecules; Step (2a) is to convert the immune complex to a soluble form by disrupting the bond between the first presenting group and the first accepting group. by releasing the first solid surface; Step (3) isolating the first target label from the immune complex based on the proximity between the first target label and the second target label. generating a reporter, the reporter being a nucleic acid, and the reporter (i) the first target ID, (ii) the second target ID, or (iii) the first target ID and the second target ID. said generating including both of Step (4) detecting the reporter, thereby detecting the analyte: The assay method comprising:

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

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

4. Step (2e): A sample label comprising a sample-specific ID ("sample ID") is added to (i) the first target label (ii) to the second target label, or (iii) to both the first target label and the second target label.

4. The assay method of claim 3, further comprising binding to:

5. 5. The method of claim 1, wherein step (4) further comprises PCR amplification of the nucleic acid reporter. Assay method.

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

7. (i) the first presenting group is a first nucleic acid tag (“first tag”) and the first accepting group is 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 The method of any one of claims 2 to 6, which is a second nucleic acid capture probe ("second probe"). Assay methods.

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 The method of any one of claims 2 to 6, which is a second nucleic acid capture probe ("second probe"). Assay methods.

9. (i) a streptomycin-based antibody in which the first probe is directly coupled to the first solid surface; conjugated to biotin, which binds to avidin or avidin; or (ii) a streptavidin-coated antibody in which the second probe is directly coupled to the second solid surface; 7 or 8, conjugated to biotin that binds to avidin or avidin. The assay method according to any one of the preceding claims.

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; mosquito; (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 of any one of claims 1 to 10, wherein the sample is a serum sample or a plasma sample. method.

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

13. The first target label is indirectly bound to the first binder, and the second target 13. The method of claim 1, wherein the label is directly bound to the second binder. Assay methods.

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

15. The first presentation group is non-covalently bound to the first target label, and the second presentation group is non-covalently bound to the first target label.

15. The method of claim 1, wherein the indicator group is non-covalently bound to the second target label. The assay method described above.

16. The sample label 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. In step (3), the first tag and the second tag are linked to form the nucleic acid receptor. a nucleic acid reporter composed of a fragment of the first tag and a fragment of the second tag; 18. The assay method of any one of claims 7 to 17, comprising detecting a target protein.

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

21. 21. Any of claims 18 to 20, wherein the nucleic acid reporter is formed by proximity ligation. The assay method according to any one of claims 1 to 4.

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

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

24. By simultaneously detecting the unique target ID associated with each analyte, the small number of analytes in the sample can be detected. The assay of any one of claims 1 to 23, comprising simultaneously detecting at least two analytes. Say method.

25. In step (4), detecting the analyte comprises detecting the first target ID and the second target I.

25. The assay method of any one of claims 1 to 24, comprising simultaneous detection of D.

26. Step (1) further comprises mixing a reference analyte, and the reference analyte is present in the sample.

26. The assay method of any one of claims 1 to 25, wherein the analyte is absent.

27. Prior to or simultaneously with the detection in step (4), nucleic acid reporters from at least two samples are pooling the nucleic acid reporters associated with each of the at least two samples; The unique sample IDs in the at least two samples are simultaneously detected to identify the respective components. The assay method of any one of claims 4 to 26, further comprising simultaneously detecting the precipitate. Law.

28. The reporter is a multiplexed qPCR, a multiplexed digital PCR, or a 28. The assay method of any one of claims 1 to 27, wherein the assay is detected by GS.

29. The detecting step comprises: detecting the reporter generated from the analyte of the sample; 29. The method of claim 26, further comprising normalizing to a reporter generated from the analyte. The assay method according to any one of claims 1 to 4.

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