WO2021113290A1 - Nucleic acid linked immune-sandwich assay (nulisa) - Google Patents

Nucleic acid linked immune-sandwich assay (nulisa) Download PDF

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Publication number
WO2021113290A1
WO2021113290A1 PCT/US2020/062791 US2020062791W WO2021113290A1 WO 2021113290 A1 WO2021113290 A1 WO 2021113290A1 US 2020062791 W US2020062791 W US 2020062791W WO 2021113290 A1 WO2021113290 A1 WO 2021113290A1
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Prior art keywords
fragment
tag
probe
nucleic acid
solid surface
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PCT/US2020/062791
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English (en)
French (fr)
Inventor
Yuling Luo
Wei FENG
Adrian GRZYBOWSKI
Yiyuan YIN
Shiping Chen
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ALAMAR BIOSCIENCES Inc
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ALAMAR BIOSCIENCES Inc
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Priority to JP2022533354A priority Critical patent/JP7727628B2/ja
Priority to EP20897209.1A priority patent/EP4069731A4/en
Priority to CN202080095593.8A priority patent/CN115066434A/zh
Priority to US17/330,331 priority patent/US12105084B2/en
Priority to US17/330,385 priority patent/US12188929B2/en
Publication of WO2021113290A1 publication Critical patent/WO2021113290A1/en
Anticipated expiration legal-status Critical
Priority to US18/764,021 priority patent/US20250138000A1/en
Priority to JP2025133045A priority patent/JP2025166110A/ja
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/247—IL-4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/248—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

  • the present invention relates to the field of molecular biology. Specifically, the present disclosures relate to highly sensitive immunoassays for detection of target biological molecules or molecular complexes.
  • Embodiment 1 An assay method for detecting an analyte in a sample, comprising:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;
  • Embodiment 2 The assay method of embodiment 1, wherein (i) the first target label comprises a first identity barcode (“ID”) that is analyte-specific (“target ID”); (ii) the second target label comprises a second target ID; or (iii) both (i) and (ii).
  • ID first identity barcode
  • target ID analyte-specific
  • target ID second target label
  • Embodiment 3 The assay method of embodiment 1 or 2, wherein (i) the reporter comprises the first target ID; (ii) the reporter comprises the second target ID; or (iii) both (i) and (ii).
  • Embodiment 4 An assay method for detecting an analyte in a sample, comprising:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first and the second target ID;
  • Embodiment 5 The assay method of embodiment 4, wherein the reporter is generated from the immunocomplex based on proximity between the first target label and the second target label.
  • Embodiment 6 The assay method of any one of embodiments 1 to 5, wherein the reporter is a nucleic acid reporter.
  • Embodiment 7 The assay method of embodiment 6, wherein:
  • the first target ID in the reporter is a complementary sequence of the first target ID in the first binding moiety
  • the second target ID in the reporter is a complementary sequence of the second target ID in the first binding moiety
  • Embodiment 8 The assay method of any one of embodiments 1 to 7, further comprising a step (2a) between step (2) and (3): releasing the immunocomplex from the first solid surface by disrupting the binding between the first presenting group and the first receiving group, wherein step (2a) is before, after or simultaneous of step (3).
  • Embodiment 9 The assay method of embodiment 8, wherein the second binding moiety further comprises a second presenting group.
  • Embodiment 10 The assay method of embodiment 9, wherein the method further comprises a step 2(b) between step 2(a) and step (3):
  • (2b) introducing a second solid surface and recapturing the immunocomplex on the second solid surface via binding between the second presenting group and the second receiving group coupled to the second solid surface.
  • Embodiment 11 The assay method of embodiment 10, wherein the method further comprises a step 2(c) between step 2(b) and step (3)
  • Embodiment 12 The assay method of embodiment 10 or 11, further comprising a step (2d): releasing the immunocomplex from the second solid surface by disrupting the binding between the second presenting group and the second receiving group.
  • Embodiment 13 The assay method of embodiment 12, wherein step (2d) is before, after or simultaneous of step (3).
  • Embodiment 14 The assay method of any one of embodiments 2 to 13, wherein (i) the first target ID and the second target ID are identical; or (ii) the first target ID and the second target ID are different.
  • Embodiment 15 The assay method of any one of the preceding embodiments, further comprising a step (2e): binding a sample label comprising an ID that is sample-specific (“sample ID”) (i) to the first target label, (ii) to the second target label, or (iii) to both the first target label and the second target label.
  • sample ID an ID that is sample-specific
  • Embodiment 16 The assay method of embodiment 15, wherein the reporter formed in each sample comprises a sample ID.
  • Embodiment 17 The assay method of any one of the preceding embodiments, wherein the first presenting group is a polypeptide fused to the first binder, a polynucleotide conjugated to the first binder, or a chemical compound conjugated to the first binder.
  • Embodiment 18 The assay method of any one of embodiments 9 to 17, wherein the second presenting group is a polypeptide fused to the second binder, a polynucleotide conjugated to the second binder, or a chemical compound conjugated to the second binder.
  • Embodiment 19 The assay method of any one of the preceding embodiments, wherein the method further comprises releasing the reporter from the immunocomplex.
  • Embodiment 20 The assay method of any one of embodiments 6 to 19, wherein step (4) further comprises PCR amplification of the nucleic acid reporter.
  • Embodiment 21 The assay method of any one of embodiments 6 to 20, wherein the method further comprises purifying the nucleic acid reporter.
  • Embodiment 22 The assay method of any one of the preceding embodiments, wherein step (1) comprises forming the immunocomplex in the solution before capturing the immunocomplex on the first solid surface.
  • Embodiment 23 The assay method of any one of embodiments 1 to 21, wherein step (1) comprises pre-capturing the first binder on the first solid surface before forming the immunocomplex on the first solid surface.
  • Embodiment 24 The assay method of any one of embodiments 1 to 21, wherein the immunocomplex is formed in the solution and captured on the first solid surface simultaneously in step (1).
  • Embodiment 25 The assay method of any one of the preceding embodiments, wherein:
  • the first binder binds to the analyte indirectly and the second binder binds to the analyte directly;
  • the first binder binds to the analyte indirectly and the second binder binds to the analyte indirectly.
  • Embodiment 26 The assay method of any one of the preceding embodiments, wherein:
  • the first binder binds to a first primary antibody or a fragment thereof that binds directly to the analyte
  • the second binder binds to a second primary antibody or a fragment thereof that binds directly to the analyte
  • Embodiment 27 The assay method of any one of embodiments 1 to 25, wherein:
  • the first and second binders bind to non-overlapping epitopes on the analyte
  • the first and second binders bind to different epitopes on the analyte.
  • Embodiment 28 The assay method of any one of the preceding embodiments, further comprising:
  • step (2) at least one additional cycle of recapture between steps (2) and (3), comprising: releasing the immunocomplex from the solid surface that it is captured on, recapturing the immunocomplex on an additional solid surface coupled with the first receiving group, and washing the additional solid surface to remove unbound molecules;
  • step (2c) at least one additional cycle of recapture between steps (2c) and (2d), comprising: releasing the immunocomplex from the solid surface that it is captured on, recapturing the immunocomplex on an additional solid surface coupled with the first or the second receiving group, and washing the additional solid surface to remove unbound molecules; or
  • Embodiment 29 The assay method of any one of embodiments 8 to 28, wherein any of the releasing is by increasing temperature to 70°C.
  • Embodiment 30 The assay method of any one of preceding embodiments, wherein (i) the first presenting group binds the first receiving group via a thioester group, a disulfide linkage, or a cleavable linkage; (ii) the second presenting group binds the second receiving group via a thioester group, a disulfide linkage, or a cleavable linkage; or both (i) and (ii).
  • Embodiment 31 The assay method of embodiment 30, wherein (i) the first presenting group binds the first receiving group via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage; (ii) the second presenting group binds the second receiving group via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage; or both (i) and (ii).
  • Embodiment 32 The assay method of any one of embodiments 1 to 29, wherein (i) the first presenting group binds the first receiving group via a protein-protein interaction; (ii) the second presenting group binds the second receiving group via a protein-protein interaction; or both (i) and (ii).
  • Embodiment 33 The assay method of any one of embodiments 1 to 29, wherein (i) the first presenting group binds the first receiving group via biotin to streptavidin or avidin; (ii) the second presenting group binds the second receiving group via biotin to streptavidin or avidin; or both (i) and (ii).
  • Embodiment 34 The assay method of any one of embodiments 1 to 29, wherein
  • the first presenting group is a first nucleic acid tag (the “first tag”) and the first receiving group is a first nucleic acid capture probe (the “first probe”);
  • Embodiment 35 The assay method of any one of embodiments 9 to 29, wherein (i) the first presenting group is a first nucleic acid tag (the “first tag”) and the first receiving group is a first nucleic acid capture probe (the “first probe”); and (ii) the second presenting group is a second nucleic acid tag (the “second tag”) and the second receiving group is a second nucleic acid capture probe (the “second probe”).
  • Embodiment 36 The assay method of embodiment 34 or 35, wherein:
  • the first probe is a protein that specifically binds to the first tag
  • the first probe is a protein and nucleic acid complex that specifically binds to the first tag
  • 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;
  • the first probe is a nucleic acid molecule, wherein the first probe or a fragment thereof hybridizes with the first tag or a fragment thereof.
  • Embodiment 37 The assay method of any one of embodiments 34 to 36, wherein:
  • the second probe is a protein that specifically binds to the second tag
  • the second probe is a protein and nucleic acid complex that specifically binds to the second tag
  • 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;
  • the second probe is a nucleic acid molecule, wherein the second probe or a fragment thereof hybridizes with the second tag or a fragment thereof.
  • Embodiment 38 The assay method of embodiment 36 or 37, wherein the complementarity is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compl ementati on .
  • Embodiment 39 The assay method of any one of embodiments 34 to 38, wherein (i) the first probe is directly coupled to the first solid surface; or (ii) the second probe is directly coupled to the second solid surface.
  • Embodiment 40 The assay method of any one of embodiments 34 to 38, wherein (i) the first probe is directly coupled to the first solid surface; and (ii) the second probe is directly coupled to the second solid surface.
  • Embodiment 41 The assay method of any one of embodiments 34 to 38, wherein (i) the first probe hybridizes with a universal probe that is directly coupled to the first solid surface; or (ii) the second probe hybridizes with a universal probe that is directly coupled to the second solid surface.
  • Embodiment 42 The assay method of any one of embodiments 34 to 38, wherein (i) the first probe hybridizes with a universal probe that is directly coupled to the first solid surface; and (ii) the second probe hybridizes with a universal probe that is directly coupled to the second solid surface.
  • Embodiment 43 The assay method of any one of embodiments 34 to 38, wherein (i) the first probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the first solid surface; or (ii) the second probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the second solid surface.
  • Embodiment 44 The assay method of any one of embodiments 34 to 38, wherein (i) the first probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the first solid surface; and (ii) the second probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the second solid surface.
  • Embodiment 45 The assay method of any one of embodiments 34 to 44, wherein the first tag and second tag are collaboratively captured on the first solid surface in step (1).
  • Embodiment 46 The assay method of embodiment 45, wherein a 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 the second tag or a fragment thereof, wherein the complementary region includes the unconjugated ends of the first and the second tags.
  • Embodiment 47 The assay method of embodiment 45, wherein a continuous fragment of the first probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first and second tags are linked to form a linked nucleic acid, a joint region of the linked nucleic acid is complementary to the continuous fragment of the first probe.
  • Embodiment 48 The assay method of embodiment 45, wherein a first fragment of the first probe is complementary to the first tag or a fragment thereof, and wherein a separate second fragment of the first probe is complementary to the second tag or a fragment thereof; and wherein the complementary region does not include the unconjugated ends of the first and the second tags.
  • Embodiment 49 The assay method of embodiment 45, wherein the first solid surface is coupled with both the first probe and an additional nucleic acid probe, and wherein the additional probe or a fragment thereof is complementary to the first tag or a fragment thereof.
  • Embodiment 50 Embodiment 50.
  • Embodiment 51 The assay method of embodiment 50, wherein a first fragment of the second probe is complementary to the first tag or a fragment thereof, and a second fragment of the second probe is complementary to the second tag or a fragment thereof, wherein the complementary region includes the unconjugated ends of the first and the second tags.
  • Embodiment 52 The assay method of embodiment 50, wherein a continuous fragment of the second probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first and second tags are linked to form a linked nucleic acid, a joint region of the linked nucleic acid is complementary to the continuous fragment of the second probe.
  • Embodiment 53 The assay method of embodiment 50, wherein a first fragment of the second probe is complementary to the first tag or a fragment thereof, and wherein a separate second fragment of the second probe is complementary to the second tag or a fragment thereof; and wherein the complementary region does not include the unconjugated ends of the first and the second tags.
  • Embodiment 54 The assay method of embodiment 50, wherein the second solid surface is coupled with both the second probe and an additional nucleic acid probe, and wherein the additional probe or a fragment thereof is complementary to the second tag or a fragment thereof.
  • Embodiment 55 The assay method of any one of embodiments 34 to 54, wherein (i) the complementary fragments of the first tag and the first probe consist of 10 to 30 base pairs; (ii) the complementary fragments of the second tag and the second probe consist of 10 to 30 base pairs; or both (i) and (ii).
  • Embodiment 56 The assay method of any one of embodiments 34 to 55, wherein (i) the complementary fragments of the first tag and the first probe consist of 20 to 30 base pairs; (ii) the complementary fragments of the second tag and the second probe consist of 20 to 30 base pairs; or both (i) and (ii)
  • Embodiment 57 The assay method of any one of embodiments 34 to 56, wherein (i) the first tag comprises an A and/or T rich sequence and the first probe comprises a complementary A and/or T rich sequence; (ii) the second tag comprises an A and/or T rich sequence and the second probe comprises a complementary A and/or T rich sequence; or both (i) and (ii).
  • Embodiment 58 The assay method of embodiment 57, wherein the A and/or T rich sequence has a short length such that the binding from complementary A and/or T rich sequence is weaker than both the binding between first binder and the analyte and the binding between the second binder and the analyte, thereby keeping immunocomplex stable in releasing step (2a) and/or (2d).
  • Embodiment 59 The assay method of any one of the preceding embodiments, wherein the analyte is a binding pair of two molecules; wherein the first binder binds one molecule of the binding pair, and the second binder binds the other molecule of the binding pair.
  • Embodiment 60 The assay method of any one of the preceding embodiments, wherein the analyte is a nucleic acid, and the first and second binders for the nucleic acid analyte comprise nucleic acids that are complementary to different fragments of the nucleic acid analyte.
  • Embodiment 61 Embodiment 61.
  • the assay method of any one of embodiments 1 or 59 wherein the analyte is a peptide or a protein, and (i) the first binder is an antibody or an antibody fragment that specifically binds the analyte; (ii) the second binder is an antibody or an antibody fragment that specifically binds the analyte; or both (i) and (ii).
  • Embodiment 62 The assay method of any one of the preceding embodiments, wherein the sample is a serum sample or a plasma sample.
  • Embodiment 63 The assay method of any one of embodiments 6 to 62, wherein:
  • step (3) the nucleic acid reporter is generated while the immunocomplex is being captured on the first solid surface
  • step (3) the nucleic acid reporter is generated after the immunocomplex is released from the first solid surface.
  • Embodiment 64 The assay method of any one of embodiments 10 to 62, wherein:
  • step (3) the nucleic acid reporter is generated while the immunocomplex is being captured on the second solid surface;
  • step (3) the nucleic acid reporter is generated after the immunocomplex is released from the second solid surface.
  • Embodiment 65 The assay method of any one of embodiments 1 to 64, wherein: (i) the first target label is directly bound to the first binder; or (ii) the first target label is indirectly bound to the first binder.
  • Embodiment 66 The assay method of any one of embodiments 1 to 65, wherein: (i) the second target label is directly bound to the second binder; or (ii) the second target label is indirectly bound to the second binder.
  • Embodiment 67 The assay method of any one of embodiments 1 to 66, wherein:
  • the first target label is non-covalently bound to the first binder
  • the first target label is conjugated to the first presenting group
  • the first target label is non-covalently bound to the first presenting group
  • the first target label is part of the first presenting group.
  • Embodiment 68 The assay method of any one of embodiments 1 to 67, wherein:
  • the second target label is non-covalently bound to the second presenting group
  • the second target label is part of the second presenting group.
  • Embodiment 69 The assay method of any one of embodiments 1 to 68, wherein: (i) the first presenting group is directly bound to the first binder; or (ii) the first presenting group is indirectly bound to the first binder.
  • Embodiment 70 The assay method of any one of embodiments 9 to 69, wherein: (i) the second presenting group is directly bound to the second binder; or (ii) the second presenting group is indirectly bound to the second binder.
  • Embodiment 71 The assay method of any one of embodiments 1 to 70, wherein:
  • the first presenting group is non-covalently bound to the first binder; (iii) the first presenting group is conjugated to the first target label;
  • the first presenting group is non-covalently bound to the first target label
  • the first presenting group is part of the first target label.
  • Embodiment 72 The assay method of any one of embodiments 9 to 71, wherein:
  • the second presenting group is non-covalently bound to the second target label
  • Embodiment 73 The assay method of any one of embodiments 1 to 72, wherein:
  • the first target label is a nucleic acid molecule
  • the second target label is a nucleic acid molecule
  • Embodiment 74 The assay method of any one of embodiments 34 to 73, wherein:
  • Embodiment 75 The assay method of any one of embodiments 15 to 74, wherein the sample label is a single-stranded nucleic acid molecule (“single-stranded sample label”).
  • Embodiment 76 The assay method of any one of embodiments 15 to 74, wherein the sample label is a double-stranded nucleic acid molecule (“double-stranded sample label”).
  • Embodiment 77 The assay method of embodiment 76, wherein the sample label is:
  • Embodiment 78 The assay method of embodiment 77, wherein the sample label:
  • Embodiment 79 The assay method of any one of embodiments 34 to 78, wherein step (3) comprises generating the nucleic acid reporter by linking:
  • a surrogate nucleic acid of the first tag (the “first surrogate”) and the second tag, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second tag;
  • Embodiment 80 The assay method of any one of embodiments 34 to 78, wherein step (3) comprises generating the nucleic acid reporter by linking:
  • a surrogate nucleic acid of the first tag (the “first surrogate”) and the second target label, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second target label;
  • Embodiment 81 The assay method of any one of embodiments 34 to 78, wherein step (3) comprises generating the nucleic acid reporter by linking:
  • a surrogate nucleic acid of the first target label (the “first surrogate”) and the second tag, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second tag;
  • Embodiment 82 The assay method of any one of embodiments 34 to 78, wherein step (3) comprises generating the nucleic acid reporter by linking:
  • a surrogate nucleic acid of the first target label (the “first surrogate”) and the second target label, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second target label;
  • Embodiment 83 The assay method of any one of embodiments 79 to 82, wherein the linking comprises linking:
  • sample surrogate a surrogate nucleic acid of the single-stranded sample label
  • sample surrogate a surrogate nucleic acid of the single-stranded sample label
  • sample surrogate a surrogate nucleic acid of the single-stranded sample label (“sample surrogate”) or one strand of the double-stranded sample label
  • sample surrogate a surrogate nucleic acid of the single-stranded sample label or one strand of the double-stranded sample label
  • sample surrogate a surrogate nucleic acid of the single-stranded sample label (“sample surrogate”) or one strand of the double-stranded sample label; wherein the sample label or a fragment thereof is complementary to the sample surrogate or a fragment thereof.
  • Embodiment 84 The assay method of embodiment 83, wherein the linking in each of (i) to (iv) comprises linking (c) between (a) and (b).
  • Embodiment 85 The assay method of any one of embodiments 79 to 84, wherein the nucleic acid reporter is formed by proximity ligation.
  • Embodiment 86 The assay method of any one of embodiments 79 to 84, wherein the nucleic acid reporter is formed by proximity extension.
  • Embodiment 87 The assay method of any one of any one of embodiments 79 to 86, wherein the nucleic acid reporter comprises (a) the first target ID or a surrogate nucleic acid of the first target ID (the “first target ID surrogate”), (b) the second target ID or a surrogate nucleic acid of the second target ID (the “second target ID surrogate”), and (c) a sample ID.
  • Embodiment 88 The assay method of any one of embodiments 2 to 87, comprising simultaneously detecting at least two analytes in the sample by simultaneously detecting the unique target IDs associated with each analyte.
  • Embodiment 89 The assay method of embodiment 88, comprising proportionally reducing the signal from at least one of the analytes, by adding a non-functional binder to the solution in step (1), wherein the non-functional binder competes with the first binder for binding to the analyte but is either unconjugated or conjugated to a presenting group that does not bind the first receiving group.
  • Embodiment 90 The assay method of any one of embodiments 2 to 89, wherein in step (4), detecting the analyte comprises the co-detection of the first and the second target IDs.
  • Embodiment 91 The assay method of any one of the preceding embodiments, wherein in step (1) further comprising mixing a reference analyte.
  • Embodiment 92 The assay method of embodiment 91, wherein the reference analyte is an analyte that is absent in the sample.
  • Embodiment 93 The assay method of embodiment 91 or 92, wherein the reference analyte is a protein, a nucleic acid, or a chemical compound that is absent in the sample.
  • Embodiment 94 The assay method of embodiment 93, wherein the reference analyte is a viral protein, a bacterial protein, or an insect protein.
  • Embodiment 95 The assay method of any one of embodiments 15 to 94, comprising simultaneously detecting the analyte in at least two samples, by simultaneously detecting the unique sample IDs in the nucleic acid reporters associated with each sample.
  • Embodiment 96 The assay method of embodiment 95, further comprising pooling the nucleic acid reporters from the at least two samples before or simultaneously of the detection in step (4).
  • Embodiment 97 The assay method of any one of embodiments 15 to 95, comprising simultaneously detecting at least two analytes in at least two samples, by simultaneously detecting the unique sample IDs and unique target IDs in the nucleic acid reporters associated with each analyte in each sample.
  • Embodiment 98 The assay method of embodiment 97, further comprising pooling the nucleic acid reporters for the at least two analytes from the at least two samples before or simultaneously of the detection in step (4).
  • Embodiment 99 The assay method of any one of the preceding embodiments, wherein the nucleic acid reporters are detected by multiplexed qPCR, multiplexed digital PCR, orNGS.
  • Embodiment 100 The assay method of any one of the preceding embodiments, wherein the nucleic acid reporters are detected by NGS.
  • Embodiment 101 The assay method of any one of embodiments 91 to 100, wherein detecting further comprises normalizing the reporters generated from the analytes of the samples against the reporter generated from the reference analyte.
  • Embodiment 102 The assay method of any one of the preceding embodiments, wherein (i) the first solid surface is selected from the group consisting of a magnetic particle surface and a well of a microtiter plate; (ii) the second solid surface is selected from the group consisting of a magnetic particle surface and a well of a microtiter plate; or both (i) and (ii). [00105]
  • Embodiment 103 An assay method for detecting an analyte in a sample, comprising:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • sample ID an ID that is sample-specific (“sample ID”) (i) to the first target label, (ii) to the second target label, or (iii) to both the first target label and the second target label;
  • nucleic acid reporter comprises the first target ID, the second target ID, and the sample ID
  • Embodiment 104 An assay method for detecting an analyte in at least two samples, comprising:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • sample ID an ID that is sample-specific (“sample ID”) (i) to the first target label, (ii) to the second target label, or (iii) to both the first target label and the second target label;
  • nucleic acid reporter comprises the first target ID, the second target ID, and the sample ID
  • NGS next generation sequencing
  • Embodiment 105 A system for detecting an analyte in a sample comprising
  • a first binding moiety comprising a first binder, a first presenting group, and a first target label
  • the first presenting group binds the first receiving groups.
  • Embodiment 106 The system of embodiment 105, wherein (i) the first target label comprises a first identity barcode (“ID”) that is analyte-specific (“target ID”); (ii) the second target label comprises a second target ID; or (iii) both (i) and (ii).
  • ID first identity barcode
  • target ID analyte-specific
  • target ID second target label
  • Embodiment 107 A system for detecting an analyte in a sample comprising
  • a first binding moiety comprising a first binder, a first presenting group, and a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”);
  • the first presenting group binds the first receiving groups.
  • Embodiment 108 The system of any one of embodiments 105 to 107, wherein the reporter is a nucleic acid reporter.
  • Embodiment 109 The system of any one of embodiments 105 to 108, wherein the second binding moiety further comprises a second presenting group, wherein the system further comprises a second receiving group, and wherein the second presenting group binds the second receiving groups.
  • Embodiment 110 The system of any one of embodiments 106 to 109, wherein (i) the first target ID and the second target ID are identical; or (ii) the first target ID and the second target ID are different.
  • Embodiment 111 The system of any one of embodiments 105 to 110, further comprising a sample label comprising an ID that is sample-specific (“sample ID”), where in the sample label binds (i) to the first target label, (ii) to the second target label, or (iii) to both the first target label and the second target label.
  • sample ID an ID that is sample-specific
  • Embodiment 112 The system of any one of embodiments 106 to 111, further comprising reagents for proximity ligation or proximity extension to generate a nucleic acid reporter comprising (i) the first target ID and the second target ID, or (ii) the first target ID, the second target ID, and the sample ID.
  • Embodiment 113 The system of any one of embodiments 105 to 112, further comprising a first solid surface.
  • Embodiment 114 The system of any one of embodiments 109 to 113, further comprising a second solid surface.
  • Embodiment 115 The system of embodiment 113 or 114, wherein (i) the first solid surface is a magnetic particle surface or a well of a microtiter plate; (ii) the second solid surface is a magnetic particle surface or a well of a microtiter plate; or both (i) and (ii).
  • Embodiment 116 The system of embodiment 114 or 115, wherein the first solid surface is coupled with the first receiving group and the second solid surface is coupled with the second receiving group.
  • Embodiment 117 The system of any one of embodiments 105 to 116, wherein the first presenting group is a polypeptide fused to the first binder, a polynucleotide conjugated to the first binder, or a chemical compound conjugated to the first binder.
  • Embodiment 118 The system of any one of embodiments 109 to 117, wherein the second presenting group is a polypeptide fused to the second binder, a polynucleotide conjugated to the second binder, or a chemical compound conjugated to the second binder.
  • Embodiment 119 The system of any one of embodiments 108 to 118, further comprising reagents for PCR amplification of the nucleic acid reporter.
  • Embodiment 120 The system of any one of embodiments 108 to 119, further comprising reagents for purifying the nucleic acid reporter.
  • Embodiment 121 The system of any one of embodiments 105 to 120, wherein:
  • the first binder binds to the analyte indirectly and the second binder binds to the analyte directly;
  • the first binder binds to the analyte indirectly and the second binder binds to the analyte indirectly.
  • Embodiment 122 The system of any one of embodiments 105 to 121, wherein:
  • the first binder binds to a first primary antibody or a fragment thereof that binds directly to the analyte
  • the second binder binds to a second primary antibody or a fragment thereof that binds directly to the analyte
  • Embodiment 123 The system of any one of embodiments 105 to 122, wherein:
  • the first and second binders bind to non-overlapping epitopes on the analyte
  • the first and second binders bind to different epitopes on the analyte.
  • Embodiment 124 The system of any one of embodiments 105 to 123, wherein (i) the first presenting group binds the first receiving group via a thioester group, a disulfide linkage, or a cleavable linkage; (ii) the second presenting group binds the second receiving group via a thioester group, a disulfide linkage, or a cleavable linkage; or both (i) and (ii).
  • Embodiment 125 The system of embodiment 124, wherein (i) the first presenting group binds the first receiving group via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage; (ii) the second presenting group binds the second receiving group via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage; or both (i) and (ii).
  • Embodiment 126 The system of any one of embodiments 105 to 123, wherein (i) the first presenting group binds the first receiving group via a protein-protein interaction; (ii) the second presenting group binds the second receiving group via a protein-protein interaction; or both (i) and (ii).
  • Embodiment 127 The system of any one of embodiments 105 to 123, wherein (i) the first presenting group binds the first receiving group via biotin to streptavidin or avidin; (ii) the second presenting group binds the second receiving group via biotin to streptavidin or avidin; or both (i) and (ii).
  • Embodiment 128 The system of any one of embodiments 105 to 123, wherein
  • the first presenting group is a first nucleic acid tag (the “first tag”) and the first receiving group is a first nucleic acid capture probe (the “first probe”),; or
  • Embodiment 129 The system of any one of embodiments 109 to 123, wherein (i) the first presenting group is a first nucleic acid tag (the “first tag”) and the first receiving group is a first nucleic acid capture probe (the “first probe”); and (ii) the second presenting group is a second nucleic acid tag (the “second tag”) and the second receiving group is a second nucleic acid capture probe (the “second probe”).
  • Embodiment 130 The system of embodiments 128 or 129, wherein:
  • the first probe is a protein that specifically binds to the first tag
  • the first probe is a protein and nucleic acid complex that specifically binds to the first tag
  • 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;
  • the first probe is a nucleic acid molecule, wherein the first probe or a fragment thereof hybridizes with the first tag or a fragment thereof.
  • Embodiment 131 The system of embodiments 128 or 130, wherein:
  • the second probe is a protein that specifically binds to the second tag
  • the second probe is a protein and nucleic acid complex that specifically binds to the second tag;
  • 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;
  • the second probe is a nucleic acid molecule, wherein the second probe or a fragment thereof hybridizes with the second tag or a fragment thereof.
  • Embodiment 132 The system of embodiments 130 or 131, wherein the complementarity is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compl ementati on .
  • Embodiment 133 The system of any one of embodiments 128 to 132, wherein (i) the first probe is directly coupled to the first solid surface; or (ii) the second probe is directly coupled to the second solid surface.
  • Embodiment 134 The system of any one of embodiments 128 to 132, wherein (i) the first probe is directly coupled to the first solid surface; and (ii) the second probe is directly coupled to the second solid surface.
  • Embodiment 135. The system of any one of embodiments 128 to 132, wherein (i) the first probe hybridizes with a universal probe that is directly coupled to the first solid surface; or (ii) the second probe hybridizes with a universal probe that is directly coupled to the second solid surface.
  • Embodiment 136 The system of any one of embodiments 128 to 132, wherein (i) the first probe hybridizes with a universal probe that is directly coupled to the first solid surface; and (ii) the second probe hybridizes with a universal probe that is directly coupled to the second solid surface.
  • Embodiment 137 The system of any one of embodiments 128 to 132, wherein (i) the first probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the first solid surface; or (ii) the second probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the second solid surface.
  • Embodiment 138 The system of any one of embodiments 128 to 132, wherein (i) the first probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the first solid surface; and (ii) the second probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the second solid surface.
  • Embodiment 139 The system of any one of embodiments 128 to 138, wherein the first tag and second tag are collaboratively captured on the first solid surface.
  • Embodiment 140 The system of embodiment 139, wherein a 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 the second tag or a fragment thereof, wherein the complementary region includes the unconjugated ends of the first and the second tags.
  • Embodiment 141 The system of embodiment 139, wherein a continuous fragment of the first probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first and second tags are linked to form a linked nucleic acid, a joint region of the linked nucleic acid is complementary to the continuous fragment of the first probe.
  • Embodiment 142 The system of embodiment 139, wherein a first fragment of the first probe is complementary to the first tag or a fragment thereof, and wherein a separate second fragment of the first probe is complementary to the second tag or a fragment thereof; and wherein the complementary region does not include the unconjugated ends of the first and the second tags.
  • Embodiment 143 The system of embodiment 139, wherein the first solid surface is coupled with both the first probe and an additional nucleic acid probe, and wherein the additional probe or a fragment thereof is complementary to the first tag or a fragment thereof.
  • Embodiment 144 The system of any one of embodiments 128 to 143, wherein the first tag and second tag are collaboratively captured on the second solid surface.
  • Embodiment 145 The system of embodiment 144, wherein a first fragment of the second probe is complementary to the first tag or a fragment thereof, and a second fragment of the second probe is complementary to the second tag or a fragment thereof, wherein the complementary region includes the unconjugated ends of the first and the second tags.
  • Embodiment 146 The system of embodiment 144, wherein a continuous fragment of the second probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first and second tags are linked to form a linked nucleic acid, a joint region of the linked nucleic acid is complementary to the continuous fragment of the second probe.
  • Embodiment 147 The system of embodiment 144, wherein a first fragment of the second probe is complementary to the first tag or a fragment thereof, and wherein a separate second fragment of the second probe is complementary to the second tag or a fragment thereof; and wherein the complementary region does not include the unconjugated ends of the first and the second tags.
  • Embodiment 148 The system of embodiment 144, wherein the second solid surface is coupled with both the second probe and an additional nucleic acid probe, and wherein the additional probe or a fragment thereof is complementary to the second tag or a fragment thereof.
  • Embodiment 149 The system of any one of embodiments 128 to 148, wherein (i) the complementary fragments of the first tag and the first probe consist of 10 to 30 base pairs; (ii) the complementary fragments of the second tag and the second probe consist of 10 to 30 base pairs; or both (i) and (ii).
  • Embodiment 150 The system of any one of embodiments 128 to 149, wherein (i) the complementary fragments of the first tag and the first probe consist of 20 to 30 base pairs; (ii) the complementary fragments of the second tag and the second probe consist of 20 to 30 base pairs; or both (i) and (ii).
  • Embodiment 151 The system of any one of embodiments 128 to 150, wherein (i) the first tag comprises an A and/or T rich sequence and the first probe comprises a complementary A and/or T rich sequence; (ii) the second tag comprises an A and/or T rich sequence and the second probe comprises a complementary A and/or T rich sequence; or both (i) and (ii).
  • Embodiment 152 The system of embodiment 151, wherein the A and/or T rich sequence has a short length such that the binding from complementary A and/or T rich sequence is weaker than both the binding between first binder and the analyte and the binding between the second binder and the analyte.
  • Embodiment 153 The system of any one of embodiments 105 to 152, wherein the analyte is a binding pair of two molecules; wherein the first binder binds one molecule of the binding pair, and the second binder binds the other molecule of the binding pair.
  • Embodiment 154 The system of any one of embodiments 105 to 153, wherein the analyte is a nucleic acid, and the first and second binders for the nucleic acid analyte comprise nucleic acids that are complementary to different fragments of the nucleic acid analyte.
  • Embodiment 155 The system of any one of embodiments 105 to 153, wherein the analyte is a peptide or a protein, and (i) the first binder is an antibody or an antibody fragment that specifically binds the analyte; (ii) the second binder is an antibody or an antibody fragment that specifically binds the analyte; or both (i) and (ii).
  • Embodiment 156 The system of any one of embodiments 105 to 155, wherein the sample is a serum sample or a plasma sample.
  • Embodiment 157 The system of any one of embodiments 105 to 156, (i) the first target label is directly bound to the first binder; or (ii) the first target label is indirectly bound to the first binder.
  • Embodiment 158 The system of any one of embodiments 105 to 157, wherein: (i) the second target label is directly bound to the second binder; or (ii) the second target label is indirectly bound to the second binder.
  • Embodiment 159 The system of any one of embodiments 105 to 158, wherein:
  • the first target label is non-covalently bound to the first binder
  • the first target label is conjugated to the first presenting group
  • the first target label is non-covalently bound to the first presenting group
  • the first target label is part of the first presenting group.
  • Embodiment 160 The system of any one of embodiments 105 to 159, wherein:
  • the second target label is non-covalently bound to the second presenting group
  • the second target label is part of the second presenting group.
  • Embodiment 161 The system of any one of embodiments 105 to 160, wherein: (i) the first presenting group is directly bound to the first binder; or (ii) the first presenting group is indirectly bound to the first binder. [00164] Embodiment 162. The system of any one of embodiments 109 to 161, wherein: (i) the second presenting group is directly bound to the second binder; or (ii) the second presenting group is indirectly bound to the second binder.
  • Embodiment 163 The system of any one of embodiments 105 to 162, wherein:
  • the first presenting group is non-covalently bound to the first target label
  • the first presenting group is part of the first target label.
  • Embodiment 164 The system of any one of embodiments 109 to 163, wherein:
  • the second presenting group is non-covalently bound to the second target label
  • Embodiment 165 The system of any one of embodiments 105 to 164, wherein:
  • the first target label is a nucleic acid molecule
  • the second target label is a nucleic acid molecule
  • Embodiment 166 The system of any one of embodiments 128 to 165, wherein:
  • Embodiment 167 The system of any one of embodiments 106 to 166, wherein the sample label is a single-stranded nucleic acid molecule (“single-stranded sample label”).
  • Embodiment 168 The system of any one of embodiments 106 to 166, wherein the sample label is a double-stranded nucleic acid molecule (“double-stranded sample label”).
  • Embodiment 169 The system of embodiment 168, wherein the sample label is:
  • Embodiment 170 The system of embodiment 169, wherein the sample label:
  • Embodiment 171 The system of any one of embodiments 106 to 170, wherein the system is capable of simultaneously detecting at least two analytes in the sample by simultaneously detecting the unique target IDs associated with each analyte.
  • Embodiment 172 The system of any one of embodiments 105 to 171, wherein the system further comprises a reference analyte.
  • Embodiment 173 The system of embodiment 172, wherein the reference analyte is an analyte that is absent in the sample.
  • Embodiment 174 The system of embodiment 172 or 173, wherein the reference analyte is a protein, a nucleic acid, or a chemical compound that is absent in the sample.
  • Embodiment 175. The system of embodiment 174, wherein the reference analyte is viral protein, a bacterial protein, or an insect protein.
  • Embodiment 176 The system of any one of embodiments 111 to 175, wherein the system is capable of simultaneously detecting the analyte in at least two samples, by simultaneously detecting the unique sample IDs in the nucleic acid reporters associated with each sample.
  • Embodiment 177 The system of any one of embodiments 110 to 176, wherein the system is capable of simultaneously detecting at least two analytes in at least two samples, by simultaneously detecting the unique sample IDs and unique target IDs in the nucleic acid reporters associated with each analyte in each sample.
  • Embodiment 178 The system of any one of embodiments 105 to 177, comprising reagents and/or machines for multiplexed qPCR, multiplexed digital PCR, or NGS to detect nucleic acid reporters.
  • Embodiment 179 The system of any one of embodiments 105 to 177, comprising reagents and/or machines for NGS to detect the nucleic acid reporters.
  • Embodiment 180 The system of any one of embodiments 105 to 179, wherein the system is contained in a kit.
  • Embodiment 181 The system of embodiment 180, wherein the kit further comprises a binding buffer, an immobilization buffer, a washing buffer, a release buffer, or any combination thereof.
  • Embodiment 182 A system for detecting an analyte in a sample comprising (1) a first binding moiety comprising a first binder, a first presenting group, and a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”); (2) a second binding moiety comprising a second binder, a second presenting group, and a second target label comprising a second target ID; (3) a first receiving group, a first solid surface, a second receiving group, and a second solid surface; (4) reagents for ligation and a sample label comprising an ID that is sample-specific (“sample ID”); and (5) reagents for quantitative PCR; wherein:
  • the first receiving group is coupled to the first solid surface and is configured to capture the first presenting group
  • the second receiving group is coupled to the second solid surface and is configured to capture the second presenting group
  • the first target label is directly or indirectly bound to the first binder and the second target label is directly or indirectly bound to the second binder;
  • the first presenting group is directly or indirectly bound to the first binder and the second presenting group is directly or indirectly bound to the second binder;
  • the sample label binds to both the first target label and the second target label.
  • Embodiment 183 The system of embodiment 182, wherein the system is contained in a kit.
  • Embodiment 184 The system of embodiment 183, wherein the kit further comprises a binding buffer, an immobilization buffer, a washing buffer, a release buffer, or any combination thereof.
  • Embodiment 185 A system for detecting an analyte in a sample comprising (1) a first binding moiety comprising a first binder, a first presenting group, and a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”); (2) a second binding moiety comprising a second binder, a second presenting group, and a second target label comprising a second target ID; (3) a first receiving group, a first solid surface, a second receiving group, and a second solid surface; and (4) reagents for ligation and a sample label comprising an ID that is sample-specific (“sample ID”); wherein:
  • the first receiving group is coupled to the first solid surface and is configured to capture the first presenting group
  • the second receiving group is coupled to the second solid surface and is configured to capture the second presenting group
  • the first target label is directly or indirectly bound to the first binder, and the second target label is directly or indirectly bound to the second binder;
  • the first presenting group is directly or indirectly bound to the first binder, and the second presenting group is directly or indirectly bound to the second binder;
  • the sample label binds to both the first target label and the second target label.
  • Embodiment 186 The system of embodiment 185, wherein the system is contained in a kit.
  • Embodiment 187 The system of embodiment 186, wherein the kit further comprises a binding buffer, an immobilization buffer, a washing buffer, a release buffer, or any combination thereof.
  • assay methods that use two capture binders.
  • assay methods for detecting an analyte in a sample comprising:
  • first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex, and wherein the immunocomplex is captured on a first solid surface in contact with the solution via binding between a first presenting group conjugated to the first binder and a first receiving group coupled to the first surface;
  • step (1) comprises forming the immunocomplex in the solution before capturing the immunocomplex on the first solid surface. In some embodiments, step (1) comprises pre-capturing the first binder on the first solid surface before forming the immunocomplex on the first solid surface. In some embodiments, the immunocomplex is formed in the solution and captured on the first solid surface simultaneously in step (1).
  • the assay methods provided herein further comprise at least one additional cycle of recapture between steps (5) and (6), comprising: releasing the immunocomplex from the solid surface that it is captured on, recapturing the immunocomplex on an additional solid surface coupled with the first or the second receiving group, and washing the additional solid surface to remove unbound molecules.
  • the first presenting group binds the first receiving group via a thioester group, a disulfide linkage, or a cleavable linkage;
  • the second presenting group binds the second receiving group via a thioester group, a disulfide linkage, or a cleavable linkage; or both (i) and (ii).
  • the first presenting group binds the first receiving group via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage;
  • the second presenting group binds the second receiving group via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage; or both (i) and (ii).
  • the first presenting group is a first nucleic acid tag (the “first tag”) and the first receiving group is a first nucleic acid capture probe (the “first probe”), wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; or (ii) the second presenting group is a second nucleic acid tag (the “second tag”) and the second receiving group is a second nucleic acid capture probe (the “second probe”), wherein the second probe or a fragment thereof is complementary to the second tag or a fragment thereof.
  • the first presenting group is the first tag and the first receiving group is the first probe; and (ii) the second presenting group is the second tag and the second receiving group is the second probe.
  • the first probe is directly coupled to the first solid surface; (ii) the second probe is directly coupled to the second solid surface; or both (i) and (ii).
  • the first probe hybridizes with a universal probe that is directed coupled to the first solid surface; (ii) the second probe hybridizes with a universal probe that is directed coupled to the second solid surface; or both (i) and (ii).
  • the first probe is conjugated with biotin, which binds the streptavidin or avidin that is directed coupled to the first solid surface;
  • the second probe is conjugated with biotin, which binds the streptavidin or avidin that is directed coupled to the second solid surface; or both (i) and (ii).
  • the first tag and second tag are collaboratively captured on the first solid surface in step (1).
  • a 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 the second tag or a fragment thereof, wherein the complementary region includes the unconjugated ends of the first and the second tags.
  • a continuous fragment of the first probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first and second tags are linked to form a linked nucleic acid, a joint region of the linked nucleic acid is complementary to the continuous fragment of the first probe.
  • a first fragment of the first probe is complementary to the first tag or a fragment thereof, and a separate second fragment of the first probe is complementary to the second tag or a fragment thereof; wherein the complementary region does not include the unconjugated ends of the first and the second tags.
  • the first solid surface is coupled with both the first probe and the second probe.
  • the complementary fragments of the first tag and the first probe consist of 10 to 25 base pairs; (ii) the complementary fragments of the second tag and the second probe consist of 10 to 25 base pairs; or both (i) and (ii).
  • assay methods using one capture binder comprising:
  • first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex; and wherein the immunocomplex is captured on a first solid surface in contact with the solution via hybridization between a first nucleic acid tag (the “first tag”) conjugated to the first binder and a first nucleic acid capture probe (the “first probe”) coupled to the first solid surface;
  • the second probe introducing a second solid surface coupled with a second nucleic acid probe (the “second probe”) and recapturing the immunocomplex on the second solid surface via hybridization between the first tag and the second probe;
  • the second probe is the same as the first probe.
  • step (1) comprises forming the immunocomplex in the solution before capturing the immunocomplex on the first solid surface. In some embodiments, step (1) comprises pre-capturing the first binder on the first solid surface before forming the immunocomplex on the first solid surface. In some embodiments, the immunocomplex is formed in the solution and captured on the first solid surface simultaneously in step (1).
  • assay methods provided herein comprise at least one additional cycle of recapture between steps (5) and (6), comprising: releasing the immunocomplex from the solid surface that it is captured on; recapturing the immunocomplex on an additional solid surface coupled with (a) the first probe, (b) the second probe, or (c) another nucleic acid probe that hybridizes with first tag; and washing the additional solid surface to remove unbound molecules.
  • the first probe is directly coupled to the first solid surface; (ii) the second probe is directly coupled to the second solid surface; or both (i) and (ii).
  • the first probe hybridizes with a universal probe that is directed coupled to the first solid surface; (ii) the second probe hybridizes with a universal probe that is directed coupled to the second solid surface; or both (i) and (ii).
  • the first probe is conjugated with biotin, which binds the streptavidin or avidin that is directed coupled to the first solid surface;
  • the second probe is conjugated with biotin, which binds the streptavidin or avidin that is directed coupled to the second solid surface; or both (i) and (ii).
  • the second binder is conjugated to a second nucleic acid tag (the “second tag”).
  • the first tag and second tag are collaboratively captured on the first solid surface in step (1).
  • a 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 the second tag or a fragment thereof; wherein the complementary region includes the unconjugated ends of the first and the second tags.
  • a continuous fragment of the first probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first and second tags are linked to form a linked nucleic acid, a joint region of the linked nucleic acid is complementary to the continuous fragment of the first probe.
  • a first fragment of the first probe is complementary to the first tag or a fragment thereof, and a separate second fragment of the first probe is complementary to the second tag or a fragment thereof; wherein the complementary region does not include the unconjugated ends of the first and the second tags.
  • the first solid surface is coupled with both the first probe and an additional nucleic acid probe, and the additional probe or a fragment thereof is complementary to the second tag or a fragment thereof.
  • the first tag and second tag are collaboratively captured on the second solid surface in step (4).
  • a first fragment of the second probe is complementary to the first tag or a fragment thereof, and a second fragment of the second probe is complementary to the second tag or a fragment thereof; wherein the complementary region includes the unconjugated ends of the first and the second tags.
  • a continuous fragment of the second probe consists of a first fragment and an immediately adjacent second fragment, the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first and second tags are linked to form a linked nucleic acid, a joint region of the linked nucleic acid is complementary to the continuous fragment of the second probe.
  • a first fragment of the second probe is complementary to the first tag or a fragment thereof, and a separate second fragment of the second probe is complementary to the second tag or a fragment thereof; wherein the complementary region does not include the unconjugated ends of the first and the second tags.
  • the second solid surface is coupled with both the second probe and an additional nucleic acid probe, and wherein the additional probe or a fragment thereof is complementary to the second tag or a fragment thereof.
  • the complementary fragments of the first tag and the first probe consist of 10 to 25 base pairs.
  • the assay methods provided herein detect an analyte in a sample, which include detecting the immunocomplex in step (6).
  • the sample is a serum sample or a plasma sample.
  • the analyte is a binding pair of two molecules; wherein the first binder binds one molecule of the binding pair, and the second binder binds the other molecule of the binding pair.
  • the first binder is an antibody or an antibody fragment that specifically binds the analyte;
  • the second binder is an antibody or an antibody fragment that specifically binds the analyte, or both (i) and (ii).
  • step (6) the immunocomplex is detected while being captured on a solid surface. In some embodiments, in step (6): the immunocomplex is detected after being released from a solid surface to a solution.
  • either the first binder or the second binder is conjugated with a detectable marker.
  • the first binder is conjugated with a detectable marker.
  • the second binder is conjugated with a detectable marker.
  • the detectable marker is a nucleic acid.
  • step (6) comprises generating a nucleic acid reporter by linking the first tag and the second tag, and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second tag.
  • step (6) comprises generating a nucleic acid reporter by linking: (a) the first tag and a surrogate nucleic acid of the second tag (the “second surrogate”), and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second surrogate; (b) a surrogate nucleic acid of the first tag (the “first surrogate”) and the second tag, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second tag; or (c) the first surrogate and the second surrogate, and detecting the nucleic acid reporter composed of a fragment of the first surrogate and a fragment of the second surrogate; wherein the first tag or a fragment thereof is complementary to the first surrogate or a fragment thereof, and the second tag or a fragment thereof is complementary to the second surrogate or a fragment thereof.
  • the nucleic acid reporter is formed by proximity ligation. In some embodiments, the nucleic acid reporter is formed by proximity extension.
  • the nucleic acid reporter is detected by qPCR, digital PCR, or next generating sequencing (NGS).
  • NGS next generating sequencing
  • the nucleic acid reporter is detected by Rolling Cycle Amplification (RCA), strand displacement amplification (SDA), Loop-Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), or a QuantiGene assay.
  • the nucleic acid reporter contains an identity barcode (“ID”) fragment that is analyte-specific (“target ID”) in the first tag or the first surrogate thereof, or in the second tag or the second surrogate thereof.
  • ID identity barcode
  • target ID analyte-specific
  • the nucleic acid reporter contains a first target ID in the first tag or the first surrogate, and a second target ID in the second tag or the second surrogate.
  • assay methods provided herein comprise simultaneously detecting at least two analytes in the sample by simultaneously detecting the unique target IDs associated with each analyte.
  • assay methods provided herein comprise proportionally reducing the signal from at least one of the analytes, by adding a non-functional binder to the solution in step (1), wherein the non-functional binder competes with the first binder for binding to the analyte but is either unconjugated or conjugated to a presenting group that does not bind the first receiving group.
  • At least one analyte is a nucleic acid
  • the first and second binders for the nucleic acid analyte comprise nucleic acids that are complementary to different fragments of the nucleic acid analyte.
  • detecting the analyte comprises the co-detection of the first and the second target IDs.
  • the analyte is a binding pair of two molecules; wherein the first binder binds one molecule of the binding pair, and the second binder binds the other molecule of the binding pair.
  • the nucleic acid reporter formed in each sample contains an ID that is sample-specific (“sample ID”), wherein the sample ID is (1) inserted between the first tag or surrogate thereof, and the second tag or surrogate thereof, (2) included in the first surrogate or the second surrogate, or (3) ligated to the first tag or surrogate thereof, or the second tag or surrogate thereof.
  • assay methods provided herein comprise simultaneously detecting the analyte in at least two samples, by simultaneously detecting the unique sample IDs in the nucleic acid reporters associated with each sample.
  • the nucleic acid reporter comprises (a) a target ID in the first tag or the first surrogate, or in the second tag or the second surrogate, and (b) a sample ID that is (1) inserted between the first tag or surrogate thereof, and the second tag or surrogate thereof, (2) included in the first surrogate or the second surrogate, or (3) ligated to the first tag or surrogate thereof, or the second tag or surrogate thereof.
  • assay methods provided herein comprise simultaneously detecting at least two analytes in at least two samples, by simultaneously detecting the unique sample IDs and unique target IDs in the nucleic acid reporters associated with each analyte in each sample.
  • the nucleic acid reporters are detected by multiplexed qPCR, multiplexed digital PCR, orNGS.
  • the first solid surface is a magnetic particle surface or a well of a microtiter plate;
  • the second solid surface is a magnetic particle surface or a well of a microtiter plate; or both (i) and (ii).
  • a sample comprising a first binder, a second binder, a first presenting group, a second presenting group, a first receiving group, and a second receiving group; wherein (i) the first and second binders bind non-interfering epitopes on the analyte, and (ii) the first and second presenting groups bind the first and second receiving groups, respectively.
  • systems provided herein further comprise a first solid surface and a second solid surface.
  • the first solid surface is a magnetic particle surface or a well of a microtiter plate;
  • the second solid surface is a magnetic particle surface or a well of a microtiter plate; or both (i) and (ii).
  • the first solid surface is coupled with the first receiving group, and the second first solid surface is coupled with the second receiving group.
  • systems provided herein further comprise a detectable marker.
  • the detectable marker is conjugated to the first binder or the second binder.
  • the first binder is conjugated to the first presenting group
  • the second binder is conjugated to the second presenting group.
  • the first presenting group binds the first receiving group via a thioester group, a disulfide linkage, or a cleavable linkage; (ii) the second presenting group binds the second receiving group via a thioester group, a disulfide linkage, or a cleavable linkage; or both (i) and (ii).
  • the first presenting group binds the first receiving group via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage;
  • the second presenting group binds the second receiving group via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage; or both (i) and (ii).
  • a sample comprising a first binder, a second binder, a first nucleic acid tag (the “first tag”), a second nucleic acid tag (the “second tag”), a first nucleic acid capture probe (the “first probe”), and a second nucleic acid capture probe (the “second probe”); wherein (i) the first and second binders bind non-interfering epitopes on the analyte; (ii) the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; and (iii) the second probe or a fragment thereof is complementary to the second tag or a fragment thereof.
  • systems provided herein further comprise a first solid surface and a second solid surface.
  • the first solid surface is a magnetic particle surface or a well of a microtiter plate;
  • the second solid surface is a magnetic particle surface or a well of a microtiter plate; or both (i) and (ii).
  • the first solid surface is coupled with the first probe, and the second first solid surface is coupled with the second probe.
  • the first binder is conjugated to the first tag
  • the second binder is conjugated to the second tag
  • the complementary fragments of the first tag and the first probe consist of 10 to 25 base pairs; (ii) the complementary fragments of the second tag and the second probe consist of 10 to 25 base pairs; or both (i) and (ii).
  • the first binder is an antibody or an antibody fragment that specifically binds the analyte
  • the second binder is an antibody or an antibody fragment that specifically binds the analyte; or both (i) and (ii).
  • a sample comprising a first binder, a second binder, a first nucleic acid tag (the “first tag”), a first nucleic acid capture probe (the “first probe”), and a second nucleic acid capture probe (the “second probe”); wherein (i) the first and second binders bind non-interfering epitopes on the analyte; (ii) the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; and (iii) the second probe or a fragment thereof is complementary to the first tag or a fragment thereof .
  • the second probe is the same as the first probe.
  • the first binder is conjugated to the first tag.
  • systems provided herein further comprise a detectable marker.
  • either the first binder or the second binder is conjugated to the detectable marker.
  • systems provided herein further comprise a second nucleic acid tag (the “second tag”).
  • the second tag is conjugated to the second binder.
  • a fragment of the first probe is complementary to the second tag or a fragment thereof.
  • systems provided herein further comprise a first solid surface and a second solid surface.
  • the first solid surface is a magnetic particle surface or a well of a microtiter plate
  • the second solid surface is a magnetic particle surface or a well of a microtiter plate
  • (1) the first solid surface is coupled with the first probe; (2) the second solid surface is coupled with the second probe; or both (1) and (2).
  • systems provided herein further comprise an additional nucleic acid probe, wherein the additional probe or a fragment thereof is complementary to the second tag or a fragment thereof.
  • systems provided herein further comprise a first solid surface and a second solid surface, wherein the first solid surface is coupled with the first probe and the additional probe, and the second solid surface is coupled with the second probe and the additional probe.
  • the first binder is an antibody or an antibody fragment that specifically binds the analyte;
  • the second binder is an antibody or an antibody fragment that specifically binds the analyte; or both (i) and (ii).
  • the complementary fragments of the first tag and the first probe consist of 10 to 25 base pairs;
  • the complementary fragments of the second tag and the second probe consist of 10 to 25 base pairs; or both (i) and (ii).
  • the system is contained in a kit.
  • the kit further comprises a binding buffer, an immobilization buffer, a washing buffer, a release buffer, or any combination thereof.
  • FIG. l is a schematic diagram of Single MOlecular Array (SIMOA) assay work flow. As shown, the technology is based on sandwich ELISA, but, at the final readout stage of the assay, molecules carrying the signal generation moiety are isolated, detected and counted one at a time.
  • LOD Limit of Detection
  • the lower Limit of Detection (LOD) of the assay is limited to the range of two to single digit fM, depending on the quality of the antibody pair used (Yeung, ./. 1mm. Meth. 437: 53-63 (2016)).
  • FIG.2 is a schematic diagram of Immuno-PCR work flow. As shown, a segment of the nucleic acid pre-conjugated to the detection antibody is used as the reporter of the immunocomplex, and PCR is used to amplify the reporter and generate a detectable signal. Despite the significant boost of signal strength resulted from the PCR amplification of reporters, the improvement to LOD has been modest, mostly by a factor of about 10 in comparison to sandwich ELISA (Potuckova, J Immu. Meth. 371 : 38-47 (2011)).
  • FIGs.3A-3C are schematic diagrams of Proximity Ligation Assay (“PLA”) (FIG.3A), Proximity Extension Assay (“PEA”) (FIG.3B) and solid phase PLA (FIG.3C).
  • PLA Proximity Ligation Assay
  • PEA Proximity Extension Assay
  • FIG.3C solid phase PLA
  • a nucleic acid reporter is generated when the two binders are in proximity so that their attached nucleic acids can be ligated (PLA; FIG.3A) or extended (PEA; FIG.3B).
  • Proximity-based detection assays also have LOD in the mid-to-low fM range.
  • solid phase PLA two binders to the target analyte are each conjugated with a nucleic acid and a third binder captures the analyte to solid surface (FIG.3C).
  • the solid phase proximity assay has demonstrated LODs in single digit fM range (Nong RY, Nature protocols , 8 (6): 1234-1249 (2013)).
  • the requirement of three non-interfering antibodies against the same target protein presents a significant challenge in assay development.
  • FIGs.4A-4D are schematic diagrams of assay methods provided herein using capture- and-release mechanisms.
  • FIG.4A depicts assay methods involving two capture binders.
  • FIG.4B depicts assay methods that involve two capture binders and use nucleic acid tags and probes for capture-and-release.
  • FIG.4C depicts assay methods involving one capture binder that utilizes the renewable bond between the first presenting group and the first receiving group.
  • FIG.4D depicts assay methods that involve one capture binder that utilizes the hybridization between the same nucleic acid tag and probe pair for capture-and-release.
  • FIGs.5A-5C are schematic diagrams of assay methods provided herein that use different nucleic acid reporter configurations.
  • the nucleic acid reporters are generated by linking the surrogate of the first tag (the “first surrogate”) to the second tag (FIG.5 A), by linking the first tag to the surrogate of the second tag (the “second surrogate”) (FIG.5B), or by linking the first surrogate to the second surrogate (FIG.5C).
  • the reporter can also be generated by extension.
  • FIGs.6A-6D are schematic diagrams of exemplary capture configurations used in the assay methods provided herein.
  • FIG.6A depicts direct capture configuration.
  • FIG.6B depicts indirect capture configuration using a universal probe.
  • FIG.6C depicts indirect capture configuration using a biotin/streptavidin pair.
  • FIG.6D depicts direct collaborative capture configuration, wherein the first and second nucleic acid tags collaboratively bind the nucleic acid capture probe.
  • FIGs.7A-7C are schematic diagrams of exemplary configurations of collaborative capture used in the assay methods provided herein.
  • FIG.7A depicts the collaborative capture wherein a continuous fragment of the probe hybridizes with a joint region of the first tag and a second tag.
  • FIG.7B depicts the collaborative capture wherein a fragment of the probe hybridizes with a fragment of the first tag and a separate fragment of the probe hybridizes with a fragment of the second tag.
  • FIG.7C depicts the collaborative capture wherein a solid surface is coupled with both the first and second probes, which capture the first and second tags, respectively.
  • FIGs.8A-8G are schematic diagrams of embodiments of NULISA immunoassays.
  • FIGs.8A-8B depict the assay methods that involve two releasable, orthogonal bonds, wherein the capture/release process are performed at least once between each binder and their respective solid surfaces.
  • FIG.8 A illustrates detection via the formation of a nucleic acid reporter using both nucleic acid tags conjugated to the binders; and
  • FIG.8B illustrates detection via iPCR of a nucleic acid molecule conjugated to the second binder.
  • FIGs.8C-8D depict the assay methods that involve one releasable and renewable bond on the first solid surface, wherein the capture/release are repeatedly performed between the first binder and more than one solid surfaces and the nucleic acid reporter.
  • FIG.8C illustrates detection via the formation of a nucleic acid reporter using both nucleic acid tags conjugated to the binders
  • FIG.8D illustrates detection via iPCR of a nucleic acid molecule conjugated to the second binder
  • FIG. 8E illustrates detection of the first target label that is conjugated to the first binder after capture and release have been conducted at both the first binder and the second binder
  • FIG. 8F illustrates detection of non-nucleic acid reporter or label
  • FIG. 8G illustrates an embodiment wherein binder 1 and binder 2 bind to a target indirectly, e.g. binder 1 and binder 2 bind to primary antibodies which bind directly to the analyte.
  • FIGs.9A-9B are schematic diagrams of assay methods provided herein that incorporate identity barcodes (ID) into the nucleic acid reporters.
  • ID identity barcodes
  • FIG.9A one ID is incorporated into one of the nucleic acid tags conjugated to one of the binders.
  • FIG.9B two IDs are separately incorporated in the two tags of both binders.
  • FIGs.10A-10B are schematic diagrams of assay methods provided herein using an indirect ID barcoding approach.
  • the nucleic acid reporter is formed by linking a first nucleic acid surrogate (the “first surrogate”), which can hybridize with the first tag, and the second tag, and one ID is incorporated into the first surrogate.
  • the nucleic acid reporter is formed by linking the first surrogate and a second nucleic acid surrogate (the “second surrogate”) that can hybridize with the second tag; and each surrogate is incorporated with an ID.
  • FIG.l 1 is a schematic diagram of multiplexing assay methods provided herein, which can detect multiple analytes in parallel by detecting the unique IDs incorporated in the tags conjugated to the binders for each analyte.
  • FIG.12 is a schematic diagram of assay methods provided herein that use unique IDs to achieve enhanced specificity. As shown, by requiring the co-detection of ID 1 and ID 2 that are incorporated into tags conjugated to Binder 1 and Binder 2, respectively, as the detection of a “true signal,” the methods reduce false positive signals associated with the detection of only ID 1 or ID 2, but not both.
  • FIG.13 is a schematic diagram of assay methods provided herein for detecting protein-protein interactions, wherein each protein in the binding pair is captured by a binder associated with a unique ID, and interaction of the two proteins are reflected by the co-detection of both IDs in a single immunocomplex.
  • FIGs.14A-14C are schematic diagrams of assay methods provided herein that incorporate sample-specific IDs (“sample IDs”) and can detect multiple samples in parallel.
  • FIG.14A depicts the incorporation of a sample ID between the first and second nucleic acid tags in forming the reporter for detection.
  • FIG.14B depicts the incorporation of a sample ID in either the first or second surrogate nucleic acid that is part of the reporter for detection.
  • FIG.14C depicts the ligation of a sample ID to the first or second nucleic acid tags or the surrogates thereof in forming the reporter for detection.
  • FIGs.15A-15J are schematic diagrams of assay methods provided herein for detecting nucleic acid analytes, including the formation of analyte-binder complex in solution (FIG.15 A), the capture of the immunocomplex to the first solid surface (FIG.15B), the release of the immunocomplex from the first surface (FIG.15C), the recapture of the immunocomplex to the second surface (FIG.15D), the reporter generation (FIG.15E);
  • FIGs 15F-15J illustrate an alternative workflow for nucleic acid detection, including formation of analyte-binder complex in the solution (FIG. 15F), capturing the analyte-binder complex to the first surface (FIG. 15G), release of the analyte-binder complex (FIG. 15H), recapturing the analyte-binder complex to the second surface (FIG. 151), and the generation of reporter (FIG. 15J).
  • FIG.16 is a schematic diagram of an exemplary NULISA immunoassay configuration. As shown, two binders for the analyte are each conjugated with a nucleic acid tag (“CP” and “L”, respectively), wherein CP is indirectly coupled to the first solid surface (paramagnetic beads 1) via a universal probe (poly T), and L is indirectly coupled to the second solid surface (paramagnetic beads 2) via streptavidin/biotin binding.
  • CP nucleic acid tag
  • a nucleic acid reporter of the immunocomplex is formed through the ligation of L, CP’s surrogate R (which hybridizes with part of CP) and a short oligo SI service as the Sample ID.
  • the Target ID (TI) is incorporated as a segment of L.
  • Connector (CNT) is a bridging probe deployed for ligation.
  • FIG.17 is the titration curve for human EGFR-Fc protein detection using a NULISA immunoassay.
  • FIGs.18A-18C depicts the basic configurations of the nucleic acid linked immunocomplex provided herein.
  • FIGs.19A-19D illustrates some additional configurations of the nucleic acid linked immunocomplex provided herein
  • FIGs.20A-20E illustrate a NULISA alternative workflow, including the formation of immunocomplex in the solution (FIG. 20A), capturing the binder 1 to the first solid surface via the first nucleic acid capture probe molecules (“CPI” or the “first probe”) (FIG. 20B), releasing of the immunocomplex from the first surface (FIG. 20C), and the generation of a nucleic acid reporter (FIG. 20D), and a comparison of signals generated when the immunocomplex is released to the solution (the right bar in each pair) or not (the left bar in each pair) (FIG. 20E).
  • FIGs.21 A-21I illustrate steps of a Multi-plex NULISA, including incubation (FIG.
  • FIG. 21C the release of immunocomplex from the first solid surface (FIG. 2 ID), the capture of immunocomplex to the second solid surface (FIG. 2 IE), the second wash (FIG. 2 IF), binding of the sample label and ligation to generate nucleic acid reporters containing two analyte-specific identity barcodes (“target ID”) and one sample-specific identity barcode(“sample ID”) (FIG, 21G), final wash and elution (FIG. 21H), and PCR amplification and detection (FIG. 211).
  • ligation products with Target ID and Sample ID can be pooled for sequencing with or without preamplification.
  • FIG.22 illustrates an exemplary configuration of NULISAseq.
  • CP can be the first presenting group (first tag)
  • the poly-T coupled to the paramagnetic beads can be the first receiving group (first probe)
  • R can be the first target label comprising the first target ID (TI)
  • L can be the second target label comprising the second target ID (TI)
  • biotinylated CP2 can be the second presenting group
  • streptavidin can be the second receiving group
  • CNT short for “connector”
  • SI can be the sample ID within the sample label (CNT)
  • paramagnetic beads 1 can be the first solid surface
  • the paramagnetic beads 2 can be the second solid surface.
  • FIGs.23 A-23B illustrate the mechanisms for the high sensitivity and multiplexing of
  • FIGs.24A-24B illustrate quantification of qPCR and sequencing of an analyte P24, including barcode assignment and qPCR results across different concentrations of P24 (FIG. 24A), and a comparison between quantification by sequencing versus qPCR (FIG. 24B).
  • FIGs.25A-25C illustrate results of a 5-plex NILISAseq, including sequencing results in each of the 5 repeating assays (FIG. 25 A), average of coefficient of variation (CV) (FIG. 25B), and limit of detection (LOD) for each assay (FIG. 25C).
  • the term “detect” or its grammatical equivalents are used broadly to include any means of determining the presence of the analyte (i.e. if it is present or not) or any form of measurement of the analyte.
  • detecting can include determining, measuring, or assessing the presence or absence or amount or location of analyte.
  • Quantitative, semi- quantitative and qualitative determinations, measurements or assessments are included. Such determinations, measurements or assessments can be relative, for example, when two or more different analytes in a sample are being detected, or absolute.
  • the term “quantifying” when used in the context of quantifying a target analyte(s) in a sample can refer to absolute or to relative quantification.
  • Absolute quantification can be accomplished by inclusion of known concentration(s) of one or more control analytes and/or referencing the detected level of the target analyte with known control analytes ( e.g ., through generation of a standard curve).
  • relative quantification can be accomplished by comparison of detected levels or amounts between two or more different target analytes to provide a relative quantification of each of the two or more different analytes, i.e., relative to each other.
  • analyte can be any substance (e.g . molecule) or entity to be detected by the assay methods provided herein.
  • the analyte is the target of the assay method provided herein.
  • the analyte can be any biomolecule or chemical compound that need to be detected, for example a peptide or protein, a nucleic acid molecule or a small molecule, including organic and inorganic molecules.
  • the analyte can be a cell or a microorganism, including a virus, or a fragment or product thereof.
  • the analyte can be any substance or entity for which a specific binder can be developed, and which is capable of simultaneously binding at least two “binders.”
  • the analytes are proteins or polypeptides.
  • analytes of interest include proteinaceous molecules such as polypeptides, proteins or prions or any molecule which contains a protein or polypeptide component, or fragments thereof.
  • the analyte is a wholly or partially proteinaceous molecule.
  • the analyte can also be a single molecule or a complex that contains two or more molecular subunits, which may or may not be covalently bound to one another, and which may be the same or different.
  • the analyte that can be detected by assay methods described herein can be a complex analyte, which can be a protein complex.
  • a complex can thus be a homo- or hetero-multimer.
  • Aggregates of molecules e.g. proteins
  • the aggregate analytes can be aggregates of the same protein or different proteins.
  • the analyte can also be a complex composed of proteins or peptides, or nucleic acid molecules such as DNA or RNA.
  • the analyte is a complex composed of both proteins and nucleic acids, e.g. regulatory factors, such as transcription factors.
  • sample can be any biological and clinical samples, included, e.g. any cell or tissue sample of an organism, or any body fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates, etc.
  • Environmental samples e.g. soil and water samples or food samples are also included.
  • the samples can be freshly prepared or prior-treated in any convenient way (e.g. for storage).
  • Representative samples thus include any material that contains a biomolecule, or any other desired or target analyte, including, for example, foods and allied products, clinical and environmental samples.
  • the sample can be a biological sample, including viral or cellular materials, including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles.
  • Such biological material comprise all types of mammalian and non mammalian animal cells, plant cells, algae including blue- green algae, fungi, bacteria, protozoa etc.
  • Representative samples also include whole blood and blood-derived products such as plasma, serum and buffy coat, blood cells, urine, faeces, cerebrospinal fluid or any other body fluids (e.g.
  • the sample can be pre-treated in any convenient or desired way to prepare for use in the method disclosed herein.
  • the sample can be treated by cell lysis or purification, isolation of the analyte, etc.
  • bind or its grammatical equivalents refer to an interaction between molecules (e.g. a binder and an analyte, or a presenting group and a receiving group) to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions.
  • a “binder,” as used herein in connection with an analyte, is any molecule or entity capable of binding to the analyte. In some embodiments, a binder binds specifically to its target analyte, namely, the binder binds to the target analyte with greater affinity than to other components in the sample.
  • the binder’s binding to the target analyte can be distinguished from that to non-target analytes in that the binder either does not bind to non-target analytes or does so negligibly or non-detectably, or any such non-specific binding, if it occurs, is at a relatively low level that can be distinguished.
  • the binding between the target analyte and its binder is typically non-covalent.
  • the binder used in methods provided herein can be covalently conjugated to a presenting group (e.g. a nucleic acid tag) without substantially abolishing the binding affinity of the binder to its target analyte.
  • the binder can be selected to have a high binding affinity for a target analyte.
  • the binder can have a binding affinity to the target analyte of at least about 10 4 M, at least about 10 6 M, or at least 10 9 M or higher.
  • the binder can be a variety of different types of molecules, so long as it exhibits the requisite binding affinity for the target analyte.
  • the binder can have a medium or even low affinity for its target analyte, e.g. , less than about 10 4 M.
  • the binder can be a large molecule.
  • the binders are antibodies, or binding fragments, derivatives or mimetics thereof.
  • they can be derived from polyclonal compositions, such that a heterogeneous population of antibodies differing by specificity are each conjugated with the same presenting group, or monoclonal compositions, in which a homogeneous population of identical antibodies that have the same specificity for the target analyte are each conjugated with the same presenting group.
  • the binder can be either a monoclonal or polyclonal antibody.
  • the binder is an antibody fragment, derivative or mimetic thereof, where these fragments, derivatives and mimetics have the requisite binding affinity for the target analyte.
  • Such antibody fragments or derivatives generally include at least the VH and VL domains of the subject antibodies, so as to retain the binding characteristics of the subject antibodies.
  • the binder is an antibody fragment that binds the analyte.
  • An antibody fragment as used herein refers to a molecule other than an intact antibody that comprises a portion of an antibody and generally an antigen-binding site.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab’)2, Fv, single chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies (e.g., camelid antibodies, alpaca antibodies), single variable domain of heavy chain antibodies (VHH), and multispecific antibodies formed from antibody fragments.
  • the binder is an Fab.
  • the binder is a scFv.
  • the binder is a single variable domain antibody.
  • the binder is an antibody mimetic.
  • An antibody mimetic can be molecules that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies.
  • the antibody mimetics are usually artificial peptides within a molar mass of about 2 to 20 kDa. Nucleic acids and small molecules are sometimes considered antibody mimetics as well.
  • Antibody mimetics known in the art include affibodies, affilins, affimers, affitins, alphabodies, anticalins, aptamers, avimers, DARPins, Fynomers, Kunitz domain peptides, monobodies, and nanoCLAMPs.
  • suitable for use as binders are polynucleic acid aptamers.
  • Polynucleic acid aptamers can be RNA oligonucleotides which can act to selectively bind proteins, much in the same manner as a receptor or antibody (Conrad et ak, Methods Enzymol. (1996), 267(Combinatorial Chemistry), 336-367).
  • the above described antibodies, fragments, derivatives and mimetics thereof can be obtained from commercial sources and/or prepared using any convenient technology, where methods of producing polyclonal antibodies, monoclonal antibodies, fragments, derivatives and mimetics thereof, including recombinant derivatives thereof, are known to those of the skill in the art ( e.g . U.S. Patent Nos. 5,851 ,829 and 5,965,371).
  • the binder can also be a lectin, a soluble cell-surface receptor or derivative thereof, an affibody or any combinatorically derived protein or peptide from phage display or ribosome display or any type of combinatorial peptide or protein library.
  • the binder can also be a ligand.
  • the ligand binder can have different sizes. In some embodiments, the ligand binder has a size from about 50 to about 10,000 daltons, from about 50 to about 5,000 daltons, or from about 100 to about 1000 daltons. In some embodiments, the ligand binder has a size of about 10,000 daltons or greater in molecular weight.
  • the binder is a small molecule that is capable of binding with the requisite affinity to the target analyte.
  • the small molecule can be a small organic molecule.
  • the small molecule can include one or more functional groups necessary for structural interaction with the target analyte, e.g. groups necessary for hydrophobic, hydrophilic, electrostatic or even covalent interactions.
  • the target analyte is a protein
  • the small molecule binder can include functional groups necessary for structural interaction with proteins, such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and typically include at least an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group.
  • the small molecule binder can also comprise a region that can be modified and/or participate in covalent linkage to a presenting group (e.g. a nucleic acid tag), without substantially adversely affecting the small molecules ability to bind to its target analyte.
  • a presenting group e.g. a nucleic acid tag
  • Small molecule binders can also comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Small molecule binders can also contain structures found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such compounds can be screened to identify those of interest.
  • the small molecule binder can also be derived from a naturally occurring or synthetic compound that can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known small molecules can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc ., to produce structural analogs.
  • the small molecule binders can be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means, i.e. a compound diversity combinatorial library. When obtained from such libraries, the small molecule binders are selected for demonstrating some desirable affinity for the protein target in a convenient binding affinity assay.
  • the assay methods provided herein use a first binder and a second binder that bind non-interfering “epitopes” of an analyte.
  • An epitope of an analyte refers to a site on the surface of an analyte to which a binder binds.
  • An epitope can be a localized region on the surface of an analyte.
  • An epitope can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains.
  • An epitope can have specific three dimensional structural characteristics and specific charge characteristics.
  • An epitope can be a continuous fragment of the analyte molecule.
  • An epitope can also be a molecule having more than one non-continuous fragments of the antigen linked together. If the analyte is a polypeptide or a protein, its epitope can include continuous or non-continuous sequence along the primary sequence of the polypeptide chain.
  • the first and the second binders used in the assay methods disclosed herein are of the same type of molecule.
  • the first and second binders can both be monoclonal antibodies that bind non-interfering epitopes of the analyte.
  • the first and the second binders can be different.
  • the first binder can be an antibody
  • the second binder can be a small molecule.
  • binding moiety when used in reference to an analyte, refers to a moiety including a molecule or a collection of more than one molecules, such that the moiety as a whole is capable of binding specifically to an analyte.
  • the binding moiety can include one or more binder, one or more target label, one or more sample label, and/or one or more presenting group.
  • the binding moiety can also include a binder, a target label, a sample label, and/or a presenting group.
  • the binding moiety can include a binder, a target label, and/or a presenting group.
  • the molecules in the binding moiety can be held together as a moiety by covalent, non- covalent, or a combination of covalent or non-covalent intermolecular interactions.
  • the molecules in the binding moiety can be held together via interactions with a molecule that is not part of the binding moiety, for example the analyte or one or more receiving groups. Additionally, the molecules in the binding moiety can be held together as a moiety via (i) intermolecular interactions among the molecules within the binding moiety and (ii) via interactions with a molecule that is not part of the binding moiety, for example the analyte or one or more receiving groups.
  • the binding moiety comprises or consists of a binder.
  • the binding moiety comprises or consists of a target label.
  • the binding moiety comprises or consists of a presenting group.
  • the binding moiety comprises or consists of a sample label. In one embodiment, the binding moiety comprises or consists of a binder and a target label. In some embodiments, the binding moiety comprises or consists of a binder and a presenting group. In certain embodiments, the binding moiety comprises or consists of a binder and a sample label. In further embodiments, the binding moiety comprises or consists of a target label and a presenting 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.
  • the binding moiety comprises or consists of a binder, a target label, and a presenting group. In some embodiments, the binding moiety comprises or consists of a binder, a target label and a sample label. In certain embodiments, the binding moiety comprises or consists of a binder, a presenting group and a sample label. In some embodiments, the binding moiety comprises or consists of a target label, a presenting group and a sample label. In other embodiments, the binding moiety comprises or consists of a binder, a target label, a presenting group, and a sample label.
  • the binding moiety comprises or consists of any one of a binder, a target label, a presenting group, and a sample label. In some embodiments, the binding moiety comprises or consists of any two of a binder, a target label, a presenting group, and a sample label, in any combination or permutation. In some embodiments, the binding moiety comprises or consists of any three of a binder, a target label, a presenting group, and a sample label, in any combination or permutation. In some embodiments, the binding moiety comprises or consists of all four of a binder, a target label, a presenting group, and a sample label.
  • presenting group and “receiving group” are used herein in reference to each other, which refer to a binding pair that can form ,a complex under appropriate conditions.
  • presenting groups can be conjugated to binders for the target analyte, and receiving groups can be coupled to a solid surface. Therefore, the binding between presenting group and receiving group allows the analyte to be captured on the solid surface.
  • the bond formed between the presenting group and receiving group is “releasable,” allowing the captured binder to be released from the solid surface.
  • the bond formed between the presenting group and receiving group is “renewable,” allowing the binder to be recaptured to another solid surface coupled with the same receiving group.
  • the binding pair of “presenting group” and “receiving group” can take a variety of forms. Examples of binding pairs of “presenting groups” and “receiving groups” include, but are not limited to, an antigen and an antibody against the antigen (including its fragments, derivatives or mimetics), a ligand and its receptor, complementary strands of nucleic acids, biotin and avidin (or streptavidin or neutravidin), lectin and carbohydrates, and vice versa.
  • binding pairs of “presenting groups” and “receiving groups” include fluorescein and anti -fluorescein, digioxigenin/anti-digioxigenin, and DNP (dinitrophenol)/anti-DNP, and vice versa.
  • binding pairs of “presenting groups” and “receiving groups” are complementary strands of nucleic acids, and are referred to as “tags” and “probes.”
  • binding pairs of “presenting groups” and “receiving groups” are antigens and antibodies, or antigens and antibody fragments.
  • target label refers to a moiety that facilitates detection and identification of a target molecule.
  • sample label refers to a moiety that facilitates detection and identification of sample origin of the target.
  • Suitable labels for target label and sample label include labels that can provide identifiers that can be correlated with the particular target or sample.
  • a common label that can be used for target label and/or sample label in the context of the present disclosure is sequences of nucleotides which can be correlated with the target or sample via sequencing.
  • a target label comprises a target ID.
  • a target label consists of a target ID.
  • the target label is the target ID.
  • a sample label comprises a sample ID.
  • a sample label consists of a sample ID.
  • the sample label is the sample ID.
  • Additional labels suitable for target label and sample label of the present disclosure include other identifiable or correlative information containing molecules, such as fluorescent molecules or the combination or sequences of fluorescent molecules, and/or colorimetric moieties or the combination or sequences of colorimetric moieties.
  • Other labels contemplated for the present disclosure include luminescent, light-scattering, radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Many labels are commercially available and can be used in the context of the invention.
  • identity barcode when used in reference with target or sample, refers to a molecule or a series of molecules that can be used to identify, directly or indirectly through the identification information contained in the molecule or the series of the molecules, the target or the sample.
  • Such an ID can be a nucleic acid molecule with a given sequence, a unique fluorescent label, a unique colorimetric label, a sequence of the fluorescent labels, a sequence of the colorimetric label, or any other molecules or combination of molecules, so long as molecules or the combination of molecules used as IDs can identify or otherwise distinguish a particular target or sample from other targets or samples and be correlated with the intended target or sample.
  • Nucleic acid molecules used as such IDs are also known as barcode sequences.
  • Such an ID can also be a further derivative molecule that contains the information derived from but is non-identical to the original ID, so long as such derived molecules or the derived information can identify or otherwise distinguish a particular target or sample from other targets or samples and be correlated with the intended target or sample.
  • a nucleic acid ID can include both the original nucleic acid barcode sequence and/or the reverse complement of the original nucleic acid barcode sequence, as both can distinguish and be correlated with the intended target or sample.
  • the barcode sequence can be any sequences, natural or non-natural, that are not present without being introduced as barcode sequences in the intended sample, the intended target, or any part of the intended sample or target, so that the barcode sequence can identify and be correlated with the sample or target.
  • a barcode sequence can be unique to a single nucleic acid species in a population or a barcode sequence can be shared by several different nucleic acid species in a population.
  • Each nucleic acid probe in a population can include different barcode sequences from all other nucleic acid probes in the population.
  • each nucleic acid probe in a population can include different barcode sequences from some or most other nucleic acid probes in a population.
  • all the reporters generated from immunocomplexes from one sample can have the same sample barcode sequence (sample ID).
  • sample IDs sample barcode sequence
  • targets generated from immunocomplexes from the same sample can have different target barcode sequences (target IDs).
  • all the reporters generated from immunocomplexes from the same sample, for the same target, and with the same binders can have the same target barcode sequences (target IDs).
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • assay methods that address the limitations of existing immunoassays and enable single molecule detection of immunocomplex through nucleic acid- based signal amplification.
  • the methods provided herein reduce background signal through a capture-and-release mechanism.
  • assay methods for detecting an analyte in a sample comprising the capture-and-release mechanism.
  • the capture-and-release mechanism is based on the hybridization and dissociation of nucleic acid pairs.
  • Assay methods provided herein use a capture-and-release mechanism to reduce non specific background signals.
  • the processes of capturing the immunocomplex onto solid surface and releasing it back to solution can be applied to different assay formats as disclosed herein (e.g. FIGs.4A-4D and FIGs.8A-8D).
  • assay methods provided herein use a capture-and-release mechanism that involves two binders, which can be captured by two receiving groups on two solid surfaces, respectively.
  • assay methods for detecting an analyte in a sample comprising the following steps:
  • the capture/release of the first binder (“Binder 1”) to/from the first solid surface (“Surface 1”) and the capture/release of the second binder (“Binder 2”) to/from the second solid surface (“Surface 2”) are achieved through two bonds between the presenting groups (“PG”) and the receiving groups (“RG”) that are bio-orthogonal (i.e. each independent and specific).
  • the bond between the first Presenting Group (“PG1”) and the first Receiving Group (“RG1”), namely, the first bond (“Bond 1”), is releasable.
  • the bond between the second Presenting Group (“PG2”) and the second Receiving Group (“RG2”) namely, the second bond (“Bond 2”)
  • the second bond is also releasable, and the immunocomplex can be detected either on Surface 2, or after being released from Surface 2.
  • Bond 2 is not releasable, and the immunocomplex can be detected on Surface 2.
  • Bond 1 is renewable, and at least one additional round of capture/release can be performed via Binder 1.
  • the immunocomplex released from Surface 2 can be recaptured by a new Surface 1 by forming another bond between PG1 on Binder 1 and RG1 on the new Surface 1.
  • Bond 2 is renewable, and at least one additional round of capture/release can be performed via Binder 2.
  • the immunocomplex released from either Surface 1 or Surface 2 can be recaptured by a new Surface 2 by forming another bond between PG2 on Binder 2 and RG2 on the new Surface 2.
  • both Bond 1 and Bond 2 are renewable, and more than one cycle of recapture can be performed Bond 1, Bond 2, or both.
  • neither Bond 1 nor Bond 2 is renewable, and only one cycle of capture/release is performed.
  • assay methods provided herein further comprise at least one additional cycle of recapture between steps (5) and (6), comprising: releasing the immunocomplex from the solid surface that it is captured on, recapturing the immunocomplex on an additional solid surface coupled with the first receiving group, and washing the additional solid surface to remove unbound molecules.
  • assay methods provided herein further comprise at least one additional cycle of recapture between steps (5) and (6), comprising: releasing the immunocomplex from the solid surface that it is captured on, recapturing the immunocomplex on an additional solid surface coupled with the second receiving group, and washing the additional solid surface to remove unbound molecules. Additional cycles of recapture can be included.
  • Releasable bond can be achieved through many different approaches known by an artisan in the field of protein immobilization.
  • the releasable bond is an attachment via thioester groups (e.g . US patent 4,284,553).
  • the releasable bond is a cleavable bond (e.g. Leriche, Bioorganic & Med. Chem. 20(2): 571-581 (2012)).
  • the releasable bond is disulfide linkages (e.g. Chan,
  • the releasable bond is photocleavable linkages (e.g. Photo-cleavable spacer, available at Integrated DNA Technologies, Inc.; Wan, PLoS ONE 13(2): e0191987 (2016)).
  • the releasable bond is a linkage that can be cleaved with appropriate enzymatic activities, including for example, phosphodiester, phospholipid, ester or b-galactose.
  • the releasable bond is a linkage that can be cleaved by chemoenzymatic reactions, such as Staphy-eSrtA pair (e.g.
  • the releasable bond is formed between arginine residues and a sorbent derivatized with 4-(oxoacetyl) phenoxyacetic acid (e.g. Duerksen-Hughes, Biochemistry, 28 (21):8530-6 (1989)).
  • the releasable bond is noncovalent bonds disrupted through binding competition (e.g.
  • renewable bond can also be achieved through many different approaches known by an artisan in the field of protein immobilization.
  • noncovalent bonds including hydrogen bonds, formed between binding pairs (e.g. antigen and antibody, ligand and receptor, complementary nucleic acids, etc) can be renewable.
  • the releasable and renewable bond can also be achieved through, for example, use of metal-affinity (e.g. Cheung, Appl. Microbiol. Biotechnol. 96, 1411— 1420 (2012)), N-halamine structures (e.g. Hui, Biomacromolecules 14 585-601 (2013)), or disulfide bonds (e.g. Boitieux, Anal. Chim. Acta 197: 229-237 (1987)).
  • the immunocomplex in step (3), is released from the first solid surface via binding competition by adding an excessive amount of either free first presenting group or free first receiving group to the solution.
  • a “free” presenting group refers to a presenting group that is unconjugated to a binder.
  • a “free” receiving group refers to a receiving group that is uncoupled to a solid surface.
  • the releasable and renewable bond is formed via nucleic acid hybridization, wherein the presenting group and the receiving group include nucleic acids that are complementary to each other.
  • the presenting group is a nucleic acid, which can bind a receiving group that is a DNA/RNA specific protein or aptamer binding partner (e.g. US5312730).
  • the receiving group is a nucleic acid, which can bind a presenting group that is a DNA/RNA specific protein or aptamer binding partner.
  • the first presenting group is a first nucleic acid tag (the “first tag”) and the first receiving group is a first nucleic acid capture probe (the “first probe”), wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof.
  • the second presenting group is a second nucleic acid tag (the “second tag”) and the second receiving group is a second nucleic acid capture probe (the “second probe”), wherein the second probe or a fragment thereof is complementary to the second tag or a fragment thereof.
  • the first presenting group is the first tag and the first receiving group is the first probe, wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof; and the second presenting group is the second tag and the second receiving group is the second probe, wherein the second probe or a fragment thereof is complementary to the second tag or a fragment thereof.
  • assay methods for detecting an analyte in a sample comprising the following steps:
  • assay methods provided herein further comprise at least one additional cycle of recapture between steps (5) and (6), comprising: releasing the immunocomplex from the solid surface that it is captured on via the dissolution of the hybridization between the second tag and the second probe, recapturing the immunocomplex on an additional solid surface coupled with the first receiving group, and washing the additional solid surface to remove unbound molecules.
  • assay methods provided herein further comprise at least one additional cycle of recapture between steps (5) and (6), comprising: releasing the immunocomplex from the solid surface that it is captured on via the dissolution of the hybridization between the first tag and the first probe, recapturing the immunocomplex on an additional solid surface coupled with the second receiving group, and washing the additional solid surface to remove unbound molecules.
  • 5.2.1.2 Capture-and-release with one capture binder [00339]
  • assay methods provided herein use a capture-and-release mechanism that involves only one binder, which is captured by the solid surface via one releasable and renewable bond, although two binders are used to form the immunocomplex.
  • the first binder is captured sequentially by two solid surfaces.
  • assay methods for detecting an analyte in a sample comprising the following steps:
  • first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex; and wherein the immunocomplex is captured on a first solid surface in contact with the solution via binding between a first presenting group conjugated to the first binder and a first receiving group coupled to the first solid surface;
  • the exact same capture/release mechanism can be repeated on the second solid surface. Accordingly, in some embodiments, the second receiving group is the same as the first receiving group.
  • the captures/releases can be achieved via the releasable and renewable bond between the first one (“Bond 1”) between the first presenting group (“PG1”) of the first binder (“Binder 1”) and the first receiving group (“RG1”) of the first solid surface (“Surface 1”).
  • the same presenting group (PG1) can then form a second bond (“Bond 2”) with the second receiving group (“RG2”) of the second solid surface (“Surface 2”).
  • RG2 can be the same as RG1.
  • Bond 2 is also releasable, and the immunocomplex can be detected either on Surface 2, or after being released from Surface 2.
  • Bond 2 is not releasable, and the immunocomplex can be detected on Surface 2.
  • Bond 1 is renewable
  • at least one additional round of capture/release can be performed via Binder 1.
  • the immunocomplex released from Surface 2 can be recaptured by a new Surface 1 by forming another bond between PG1 on Binder 1 and RG1 on the new Surface 1.
  • Bond 2 is also renewable, and the immunocomplex released from Surface 1 or 2 can be recaptured by a new Surface 2 by forming another bond between PG1 on Binder 1 and RG2 on the new Surface 2.
  • both Bond 1 and Bond 2 are renewable, and more than one cycle of capture/release can be performed via Bond 1, Bond 2, or both.
  • assay methods provided herein further comprise at least one additional cycle of recapture between steps (5) and (6), comprising: releasing the immunocomplex from the solid surface that it is captured on, recapturing the immunocomplex on an additional solid surface coupled with the first receiving group, and washing the additional solid surface to remove unbound molecules.
  • assay methods provided herein further comprise at least one additional cycle of recapture between steps (5) and (6), comprising: releasing the immunocomplex from the solid surface that it is captured on, recapturing the immunocomplex on an additional solid surface coupled with the second receiving group, and washing the additional solid surface to remove unbound molecules. Additional cycles of recapture can be included.
  • releasable bond and renewable bond can be achieved through many different approaches known by an artisan in the field of protein immobilization, including those disclosed herein.
  • the releasable and renewable bond is formed via nucleic acid hybridization, wherein the presenting group and the receiving group include nucleic acids that are complementary to each other.
  • the first presenting group is a nucleic acid tag (the “first tag”) and the first receiving group is a nucleic acid capture probe (the “first probe”), wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof.
  • analyte in a sample comprising: (1) mixing a first binder, a second binder, and the sample in a solution; wherein the first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex; and wherein the immunocomplex is captured on a first solid surface in contact with the solution via hybridization between a first nucleic acid tag (the “first tag”) conjugated to the first binder and a first nucleic acid capture probe (the “first probe”) coupled to the first solid surface;
  • the second probe introducing a second solid surface coupled with a second nucleic acid probe (the “second probe”) and recapturing the immunocomplex on the second solid surface via hybridization between the first tag and the second probe;
  • the second probe is the same as the first probe.
  • assay methods provided herein further comprise at least one additional cycle of recapture between steps (5) and (6), comprising: releasing the immunocomplex from the solid surface that it is captured on; recapturing the immunocomplex on an additional solid surface coupled with the probe, which can be either the first probe, the second probe, or another nucleic acid probe that hybridizes with the first tag; and washing the additional solid surface to remove unbound molecules.
  • nucleic acid hybridization brings several advantages to the assay methods disclosed herein, at least because that the conditions of nucleic acid hybridization is quite different from that of protein binding.
  • binding to target analyte by the first binder and the second binder can be conducted in solution which enables fast binding kinetics and allow large volume of sample input.
  • capture of the immunocomplex to solid surface through nucleic acid hybridization is generally more efficient, specific and predictable.
  • the sequence of capture probe can be designed to specifically targeting an intended immunocomplex, which is useful in multiplexed assay formats.
  • the dissociation of the hybridization as the release mechanism can be realized through the change of salt concentration in buffer, which helps to maximize the efficiency of the release while maintaining the integrity of the immunocomplex.
  • an appropriate release buffer condition can be identified in which the designated hybridization link dissociates to release the immunocomplex, while the immunocomplex and other hybridizations between longer or stronger complementary sequences remain stable.
  • capture/release through hybridization can be renewed and repeated multiple cycles without deterioration of efficiency and selectivity. Therefore, additional rounds of capture/release can be implemented in the assay until the desired level of low background is reached.
  • the robust capture/release mechanism ensures the unprecedented predictability and reliability of the assay methods.
  • nucleic acid capture probes coupled to the first and second surfaces help further reduce non-specific entrapment of the first binder and the second binder due to their negatively charged oligonucleotide tags.
  • the assay methods disclosed herein include step (1): formation of an immunocomplex by mixing a first binder, a second binder, and a sample in a solution, wherein the first and second binders bind non-interfering epitopes on the analyte, and wherein the immunocomplex is captured on a first solid surface in contact with the solution via the binding between a first presenting group conjugated to the first binder and a first receiving group coupled to the first surface.
  • the first presenting group is a nucleic acid tag
  • the first receiving group is a nucleic acid capture probe, wherein the probe or a fragment thereof is complementary to the tag or a fragment thereof.
  • the binders used in the assay methods can be any molecule or a portion of a molecule which binds a specific target analyte.
  • a binder can comprise any protein, peptide, nucleic acid, carbohydrate, lipid, or small molecule.
  • a binder comprises an antibody.
  • a binder comprises an antibody fragment.
  • a binder comprises an antibody mimetic.
  • a binder comprises a small molecule.
  • the binders used in assay methods disclosed herein can be conjugated to presenting groups (e.g . nucleic acid tags).
  • the binder and presenting group can be joined together either directly through a bond or indirectly through a linking group.
  • linking groups can be chosen to provide for covalent attachment of the presenting groups and binders, as well as to maintain the desired binding affinity of the binder for its target analyte.
  • Linking groups can vary depending on the binder.
  • the linking group when present, is typically biologically inert. A variety of linking groups are known to those of skill in the art and can be used in the assay methods disclosed herein.
  • a linking group comprises a spacer group terminated at either end with a reactive functionality capable of covalently bonding to the presenting group or the binder.
  • Spacer groups can include aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms such as oxygen (ethers such aspolyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that can contain heteroatoms. Spacer groups can also comprise ligands that bind to metals such that the presence of a metal ion coordinates two or more ligands to form a complex.
  • Specific spacer elements include: 1,4- diaminohexane, xylylenediamine, terephthalic acid, 3,6- dioxaoctanedioic acid, ethylenediamine- N,N-diacetic acid, 1 ,l'-ethylenebis(5-oxo-3- pyrrolidinecarboxylic acid), 4,4'- ethylenedipiperidine.
  • Potential reactive functionalities include nucleophilic functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, or binding to metals.
  • Specific examples include primary and secondary amines, hydroxamic acids, N-hydroxysuccinimidyl esters, N- hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters, glycidyl ethers, vinyl sulfones, and maleimides.
  • linker groups that can be used herein also include heterofunctional compounds, such as azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]- 3'- [2'-pyridyldithio]propionamid), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N-maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl- 4-azidobenzoate, N-succinimidyl [4-azidophenyl]-l ,3'- dithiopropionate, N-succinimidyl [4- iodoacetyljami nobenzoate, glutaraldehyde, and succinimidyl-4-[N- maleimidomethyl]cyclohexane-l-carboxylate, 3-
  • the binder/presenting group conjugates employed in the assay methods disclosed herein can be prepared using any methods known in the art.
  • the presenting groups e.g. nucleic acid tags
  • the binder can be conjugated to the binder, either directly or through a linking group.
  • the components can be covalently bound to one another through functional groups, as is known in the art, where such functional groups can be present on the components or introduced onto the components using one or more steps, e.g. oxidation reactions, reduction reactions, cleavage reactions and the like.
  • Functional groups that can be used in covalently bonding the components together include: hydroxy, sulfhydryl, amino, and the like.
  • the particular portion of the different components that are modified to provide for covalent linkage can be chosen so as not to substantially adversely interfere with that component’s desired binding affinity for the target analyte.
  • certain moieties on the components can be protected using blocking groups, as is known in the art, see e.g. Green & Wuts, Protective Groups in Organic Synthesis (John Wley & Sons) (1991); U.S. Patent No. 5,733,523.
  • the binder/presenting group conjugates can also be produced using in vitro protocols that yield nucleic acid-protein conjugates.
  • in vitro protocols of interest include: RepA based protocols (see e.g, Fitzgerald, Drug Discov. Today (2000) 5:253-258 and WO 98/37186), ribosome display based protocols (see e.g., Hanes et ak, Proc. Natl Acad. Sci.
  • the receiving groups can be “coupled” to solid surfaces by any means known in the art, which can be either direct or indirect e.g. via a linking group as described above for linking the presenting groups and the binders.
  • the receiving groups can be coupled to solid surfaces by covalent linkage (e.g. chemical cross-linking) or by non-covalent association e.g, via streptavidin-biotin based coupling (biotin being provided on one domain and streptavidin on the other).
  • the solid surface is the surface of a magnetic bead, which can be coupled with a nucleic acid capture probe as the presenting group via, for example, the binding between the carboxylic acid on the magnetic bead and the nucleic acid capture probe.
  • the solid surface can be covalently coupled with protein A/G as the presenting group.
  • the presenting groups and receiving groups can be any binding pairs disclosed herein or otherwise known in the art, which include, but are not limited to, an antigen and an antibody against the antigen (including its fragments, derivatives or mimetics), a ligand and its receptor, complementary strands of nucleic acids, biotin and avidin (or streptavidin or neutravidin), lectin and carbohydrates, and vice versa.
  • Additional binding pairs of “presenting groups” and “receiving groups” include fluorescein and anti-fluorescein, digioxigenin/anti-digioxigenin, and DNP (dinitrophenol)/anti-DNP, and vice versa.
  • binding pairs of “presenting groups” and “receiving groups” are complementary strands of nucleic acids, and are referred to as “tags” and “probes.” In some embodiments, binding pairs of “presenting groups” and “receiving groups” are antigens and antibodies, or antigens and antibody fragments.
  • the sample that can be assayed in the assay methods disclosed herein can be a material or mixture of materials containing one or more components of interest.
  • the sample is derived from a biological source.
  • the sample can be obtained from a subject, which can be a biological tissue or fluid, obtained, reached, or collected in vivo or in situ.
  • Exemplary samples include a biological fluid, such as a blood sample, a urine sample, a plasma sample, a saliva sample, a cerebrospinal fluid sample, a semen sample, a sputum sample, a mucus sample, a dialysis fluid sample, an intestinal fluid sample, a synovial fluid sample, a serous fluid sample.
  • the sample is a blood sample. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a saliva sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a cerebrospinal fluid sample. Exemplary samples include a tissue sample.
  • the tissue sample can be a liquid tissue sample.
  • the tissue sample can be a homogenized tissue sample.
  • the tissue sample can be obtained from a diseased tissue. In some embodiments, the sample is a cancer sample.
  • the solid surface can also include any support known in the art on which can be used for immobilization of molecules.
  • the solid surface can be any surfaces suitable of attaching nucleic acid and facilitates the assay step.
  • Examples of solid surfaces include beads (e.g ., magnetic beads, xMAP ® beads), particles, colloids, single surfaces, tubes, chips, multiwell plates, microtiter plates, slides, membranes, cuvettes, gels, and resins.
  • Exemplary solid surfaces can include surfaces of magnetic particles, and wells of microtiter plates. When the solid phase is a particulate material (e.g., beads), it can be distributed in the wells of multi-well plates to allow for parallel processing.
  • the solid surface is the surface of a magnetic bead.
  • the magnetic beads can be coupled with a presenting group.
  • the magnet beads can be carboxylate-modified magnetic beads, amine-blocked magnetic beads, 01igo(dT)-coated magnetic beads, streptavidin-coated magnetic beads, Protein A/G coated magnetic beads, or silica-coated magnetic beads.
  • the solid surface is a well of a microtiter plate.
  • the first and second solid surfaces are the same.
  • the first and the second solid surfaces are different.
  • both the first and second solid surfaces used in the assay methods disclosed herein are surfaces of magnetic particles.
  • both the first and second surfaces used in the assay methods disclosed herein are surfaces of microtiter plates.
  • the analyte measured in assay methods disclosed herein can be any biological molecule.
  • the analyte is a protein analyte.
  • the analyte is a peptide analyte.
  • the analyte is a complex that includes at least two molecules.
  • the analyte is a protein complex that includes at least two proteins.
  • the analyte is a binding pair of two proteins.
  • the analyte is a macromolecular complex that includes at least a protein and at least a nucleic acid.
  • the analyte is a nucleic acid analyte.
  • the analyte is a binding pair of two different molecules, the first binder binds one molecule of the binding pair, and the second binder binds the other molecule of the binding pair.
  • the analyte is a binding pair of two different proteins, the first binder binds one protein of the binding pair, and the second binder binds the other protein of the binding pair. Accordingly, provided herein are assay methods for detecting protein-protein interaction in a sample comprising the following steps:
  • the first presenting group is a first nucleic acid tag (the “first tag”) and the first receiving group is a first nucleic acid capture probe (the “first probe”), wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof.
  • the second presenting group is a second nucleic acid tag (the “second tag”) and the second receiving group is a second nucleic acid capture probe (the “second probe”), wherein the second probe or a fragment thereof is complementary to the second tag or a fragment thereof.
  • assay methods for detecting protein- protein interaction in a sample comprising the following steps:
  • the first presenting group is a first nucleic acid tag (the “first tag”) and the first receiving group is a first nucleic acid capture probe (the “first probe”), wherein the probe or a fragment thereof is complementary to the tag or a fragment thereof.
  • the second receiving group is a second nucleic acid capture probe (the “second probe”), wherein the probe or a fragment thereof is complementary to the tag or a fragment thereof.
  • the analyte can be a nucleic acid molecule (e.g . DNA & RNA).
  • the analyte is a DNA molecule.
  • the analyte is a RNA molecule.
  • Assay methods provided herein can detect nucleic acid molecules directly in samples such as plasma and urine without the need for nucleic acid isolation. As shown in FIG.15, nucleic acid analyte can be hybridized and captured onto the first surface, released into solution, and recaptured on the second surface while the target-probe complex remains intact throughout the assay procedure. Accordingly, in some embodiments, assay methods provided herein can be used for detecting a nucleic acid analyte. For example, provided herein are assay methods for detecting a nucleic acid analyte in a sample comprising the following steps:
  • first and second binders bind non-interfering epitopes on the nucleic acid analyte and form an immunocomplex, and wherein the immunocomplex is captured on a first solid surface in contact with the solution via binding between a first presenting group conjugated to the first binder and a first receiving group coupled to the first surface; wherein the first and second binders for the nucleic acid analyte are nucleic acids that are complementary to different fragments of the nucleic acid analyte;
  • assay methods for detecting a nucleic acid analyte in a sample comprising the following steps:
  • first tag a first nucleic acid tag
  • second tag a second nucleic acid tag
  • the immunocomplex is captured on a first solid surface in contact with the solution via hybridization between the first tag and a first nucleic acid capture probe (the “first probe”) coupled to the first solid surface, wherein the first probe or a fragment thereof is complementary to the first tag or a fragment thereof;
  • the methods can be used for high throughput analysis of a large number of targets by Next Generation Sequencing (NGS), including analysis of mutation, methylation, translocation, fusion, and/or copy number variation, etc.
  • NGS Next Generation Sequencing
  • selected targets can be analyzed by qPCR, digital PCR or other nucleic acid analysis technologies.
  • assay methods for detecting an analyte in a sample comprising:
  • first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex; and wherein the immunocomplex is captured on a first solid surface in contact with the solution via hybridization between a first nucleic acid tag (the “first tag”) conjugated to the first binder and a first nucleic acid capture probe (the “first probe”) coupled to the first solid surface;
  • the second probe introducing a second solid surface coupled with a second nucleic acid probe (the “second probe”) and recapturing the immunocomplex on the second solid surface via hybridization between the first tag and the second probe;
  • collaborative capture can be used in the assay methods described herein.
  • the capture probe hybridizes to a shorter segment of the nucleic acid tags of Binder 1 and 2, simultaneously.
  • the assay conditions can be set such that tags of individual binders cannot be stably hybridized to the capture probe alone due to the relatively short complementary segments.
  • the tags of the two binders are hybridized to the capture probe collaboratively with sufficient strength to be stably captured to surface. This approach further reduces non-specific binders that are not part of immunocomplex, which reduces or even eliminates the need for release/re-capture rounds.
  • direct collaborative capture is used in the assay methods provided herein, wherein the first tag and second tag are collaboratively captured on the solid surface in step (1) (FIG.6D).
  • assay methods for detecting an analyte in a sample comprising the following steps:
  • first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex
  • the immunocomplex is captured on a solid surface in contact with the solution via hybridization between a first nucleic acid tag (the “first tag”) conjugated to the first binder and a nucleic acid capture probe (“the probe”) coupled to the surface and between a second nucleic acid tag (the “second tag”) conjugated to the second binder and the probe;
  • the collaborative capture can also be repeated to further reduce nonspecific binding.
  • the immunocomplex is released from the first solid surface by dissolving the collaborative hybridization between the first and second tags and the probe and is collaboratively recaptured on the second solid surface, and wherein the second solid surface is also coupled with the probe.
  • a continuous fragment of the second probe consists of a first fragment and an immediately adjacent second fragment, wherein the first fragment is complementary to the first tag or a fragment thereof, and the second fragment is complementary to the second tag or a fragment thereof, such that when the first and second tags are linked to form a linked nucleic acid, a joint region of the linked nucleic acid is complementary to the continuous fragment of the second probe.
  • the second probe is the same as the first probe.
  • the immunocomplex is captured on the second solid surface via a different mechanism.
  • the second surface can be coupled with an antibody that binds the first binder or the second binder.
  • the complementary region includes the unconjugated ends, or “free” ends, of the first and the second tags, namely, the ends that are not conjugated with the respective binders (FIG.6D). In some embodiments, the complementary region does not include the unconjugated ends of the first and the second tags (FIG.7B). In some embodiments, the complementary region includes the conjugated ends of the first and the second tags (FIG.7B). In some embodiments, the second probe is the same as the first probe. In some embodiments, the immunocomplex is captured on the second solid surface via a different mechanism. For example, the second surface can be coupled with an antibody that binds the first binder or the second binder.
  • direct collaborative capture is used in the assay methods provided herein wherein the first solid surface is coupled with both the first probe and the second probe (FIG.7C).
  • assay methods for detecting an analyte in a sample comprising the following steps:
  • the first tag and second tag are also collaboratively recaptured on the second solid surface, and wherein the second solid surface is also coupled with both the first probe and the second probe.
  • the first solid surface is coupled with a first probe and an additional probe
  • the second solid surface is coupled with a second probe an additional probe, wherein both the first and second probes can hybridize with the first tag and the additional probe and hybridize with the second tag.
  • the second probe is the same as the first probe.
  • the immunocomplex is captured on the second solid surface via a different mechanism.
  • the second surface can be coupled with an antibody that binds the first binder or the second binder.
  • sequences between a nucleic acid tag and a nucleic acid capture probe are designed to facilitate the specific hybridization between the tag to the probe as well as the later dissociation of the hybridized immunocomplex.
  • sequences with low or no G/C can be used which, with appropriate length, can provide sufficient strength in hybridization, and which can be dissolved under low salt condition during release without disruption to the immunocomplex.
  • sequences that only include poly T/A or poly “TA”/“AT” can be used. As a person of ordinary skill in the art would understand, any sequence with appropriate hybridization strength can be utilized.
  • more than 80% of the complementary fragments of the first tag and the first probe are A and T pairs, and more than 80% of the complementary fragments of the second tag and the second probe are A and T pairs.
  • complementary fragments of the first tag and the first probe comprise A and T pairs, and complementary fragments of the second tag and the second probe comprise A and T pairs.
  • more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of the complementary fragments of a universal probe and a nucleic acid capture probe used in the assay methods provided herein are adenine (“A”) and thymine (“T”) pairs.
  • more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of the complementary fragments of the universal probe and the first probe are A and T pairs.
  • more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of the complementary fragments of the universal probe and the second capture probe are A and T pairs.
  • more than 80% of the complementary fragments of the universal probe and the first probe are A and T pairs, and more than 80% of the complementary fragments of the universal probe and the second probe are A and T pairs.
  • complementary fragments of the universal probe and the first probe comprise A and T pairs, and complementary fragments of the universal probe and the second probe comprise A and T pairs.
  • the complementary fragments of a nucleic acid tag and a nucleic acid capture probe used in the assay methods provided herein consist of 10 to 30 base pairs, 10 to 25 base pairs, 12 to 20 base pairs, or 10 to 16 base pairs. In some embodiments, the complementary fragments of a nucleic acid tag and a nucleic acid capture probe used in the assay methods provided herein consist of 10 to 25 base pairs. In some embodiments, the complementary fragments of a nucleic acid tag and a nucleic acid capture probe used in the assay methods provided herein consist of 12 to 20 base pairs.
  • the complementary fragments of a nucleic acid tag and a nucleic acid capture probe used in the assay methods provided herein consist of 12 to 16 base pairs. In some embodiments, the complementary fragments of a nucleic acid tag and a nucleic acid capture probe used in the assay methods provided herein consist of 12 to 14 base pairs. In some embodiments, the complementary fragments of the first tag and the first probe consist of 10 to 25 base pairs. In some embodiments, the complementary fragments of the second tag and the second probe consist of 10 to 25 base pairs. In some embodiments, the complementary fragments of the first tag and the first probe consist of 12 to 20 base pairs. In some embodiments, the complementary fragments of the second tag and the second probe consist of 12 to 20 base pairs. In some embodiments, the complementary fragments of the first tag and the first probe consist of 12 to 16 base pairs. In some embodiments, the complementary fragments of the second tag and the second probe consist of 12 to 16 base pairs.
  • the assay methods provided herein include detecting the second binder of the immunocomplex. In some embodiments, the assay methods provided herein include detecting the first presenting group that is conjugated to the first binder of the immunocomplex. In some embodiments, the assay methods provided herein include detecting the second presenting group that is conjugated to either the first binder or the second binder of the immunocomplex. In some embodiments, the immunocomplex is detected via a detectable marker conjugated to the first binder. In some embodiments, the immunocomplex is detected via a detectable marker conjugated to the second binder.
  • the immunocomplex can be detected by any methods known in the art.
  • the detection of immunocomplex can be achieved using antibodies which specifically bind to the first binder, the second binder, the first presenting group, or the second presenting group.
  • the antibodies for detection can be labeled with an enzyme, including for example, horseradish peroxidase, alkaline phosphatase, or beta-galactosidase, which is capable of converting a colorless or nearly colorless substrate or co-substrate into a highly colored product or a product capable of forming a colored complex with a chromogen.
  • the detection system can employ an enzyme which, in the presence of the proper substrate(s), emits light.
  • the amount of product formed can be detected either visually, spectrophotometrically, electrochemically, fluorescently or luminometrically, and can be compared to a similarly treated control.
  • the detection system can also employ radioactively labeled antibodies, in which case the amount of immune complex is quantified by scintillation counting or gamma counting.
  • Other detection systems which may be used include those based on the use of protein A derived from Staphylococcus aureus Cowan strain I, protein G from group C Staphylococcus sp. (strain 26RP66).
  • the immunocomplex is detected by immunofluorescence.
  • the immunocomplex is detected by measuring a detectable marker conjugated to a binder.
  • the detectable marker can be a fluorescent-labeled agent.
  • the labeled agent can be a secondary antibody.
  • the detectable marker can be a colorimetric detection reagent, a fluorescent detection reagent, or a chemiluminescent detection reagent.
  • the colorimetric detectable marker can include PNPP (p-nitrophenyl phosphate), ABTS (2,2'-azino- bis(3-ethylbenzothiazoline-6-sulphonic acid)) or OPD (o-phenylenediamine).
  • the secondary antibody can be derived from any mammalian organism, including mice, rats, hamsters, goats, camels, chicken, rabbit, and others.
  • the secondary antibody can also be recombinant.
  • Secondary antibodies can be conjugated to enzymes (e.g, horseradish peroxidase (HRP), alkaline phosphatase (AP), luciferase, and the like) or dyes (e.g, colorimetric dyes, fluorescent dyes, fluorescence resonance energy transfer (FRET)-dyes, time-resolved (TR)-FRET dyes, and the like).
  • the secondary antibody can be conjugated to a fluorescein (FITC) based dye, such as fluorescein isothiocyanate.
  • the secondary antibody can be conjugated to Alexa Fluor® 488 (Life technologies).
  • nucleic acid tags can be conjugated to the binders used in the assay methods disclosed herein.
  • the nucleic acid tags can be used for generation of nucleic acid reporters that allows highly sensitive detection.
  • two-capture binder assay methods for detecting an analyte in a sample comprising:
  • first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex; and wherein the immunocomplex is captured on a first solid surface in contact with the solution via hybridization between a first nucleic acid tag (the “first tag”) conjugated to the first binder and a first nucleic acid capture probe (the “first probe”) coupled to the first solid surface;
  • the second binder is also conjugated to a second nucleic acid tag (the “second tag”).
  • a nucleic acid reporter can be generated using the first tag and the second tag.
  • the nucleic acid reporters can take various forms. For example, as depicted in FIG.4B(e), the first tag and the second tag can be ligated to generate the nucleic acid reporter.
  • any of the reporter generation methods disclosed herein or otherwise known in the art can be deployed in this step, including such as ligation, polymerization extension or collaborative hybridization.
  • 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, 174,366, W09700446, Greenwood C , Biomol. Det. & Quart. 4 (2015) 10-16).
  • Proximity -based detection differ from immuno-PCR in that they depend on the simultaneous recognition of target analyte by two nucleic acid-conjugated binders in order to trigger the formation of amplifiable products.
  • the nucleic acid reporters can be generated using the nucleic acid tags in step (1) of the assay methods provided herein. As shown in FIG.4B(e), the reporter can be generated by linking the first tag and the second tag together directly via proximity ligation, proximity extension, or collaborative hybridization. Alternatively, as shown in FIGs.5A-5C, the nucleic acid reporters can be generated using nucleic acid surrogates that can hybridize with the nucleic acid tags. For example, in some embodiments, surrogate nucleic acids are used, wherein the first surrogate or a fragment thereof is complementary to the first tag or a fragment thereof, and the second surrogate or a fragment thereof is complementary to the second tag or a fragment thereof.
  • the reporter can be generated by linking the first surrogate to the second tag via proximity ligation, proximity extension, or collaborative hybridization (FIG.5 A). In some embodiments, the reporter can be generated by linking the first tag to the second surrogate via proximity ligation, proximity extension, or collaborative hybridization (FIG.5B). Alternatively, the reporter can be generated by linking the first surrogate with the second surrogate via proximity ligation, proximity extension, or collaborative hybridization (FIG.5C).
  • the first surrogate and/or the second surrogate can be added after the immunocomplex is formed.
  • the first surrogate and the second surrogate are pre-hybridized to the first tag and the second tag, respectively, prior to the immunocomplex formation.
  • step (6) of the assay methods provided herein comprises generating a nucleic acid reporter by linking the first tag and the second tag by proximity ligation, proximity extension, or collaborative hybridization, and detecting the nucleic acid reporter composed of a fragment of the first tag and a fragment of the second tag.
  • surrogate nucleic acids are used, wherein the first tag or a fragment thereof is complementary to the first surrogate or a fragment thereof, and the second tag or a fragment thereof is complementary to the second surrogate or a fragment thereof.
  • FIGs.5A-5C and 8A-8B use proximity ligation, as described above, proximity extension, collaborative hybridization, or other methods known in the art can also be used for generating the nucleic acid reporter for detection.
  • the reporters generated in the last step can be detected using any existing nucleic acid detection technologies, which include, but are not limited to, PCR, quantitative PCR (qPCR), digital PCR (dPCR) or next-generation sequencing (NGS).
  • the detection is qualitative detection.
  • the detection is quantitative detection.
  • the nucleic acid reporter is detected by qPCR.
  • the nucleic acid reporter is detected by dPCR.
  • the nucleic acid reporter is detected by NGS.
  • nucleic acid amplification/detection methods can also be used, which include, but not limited to, Rolling Cycle Amplification (RCA), strand displacement amplification (SDA), Loop-Mediated Isothermal Amplification (LAMP) and Recombinase Polymerase Amplification (RPA). Additional technologies capable of highly sensitive nucleic acid detection without the need for target amplification can also be adopted for the detection of nucleic acid reporters in the assay methods disclosed herein.
  • RCA Rolling Cycle Amplification
  • SDA strand displacement amplification
  • LAMP Loop-Mediated Isothermal Amplification
  • RPA Recombinase Polymerase Amplification
  • the nucleic acid reporter is detected by Rolling Cycle Amplification (RCA), strand displacement amplification (SDA), Loop-Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), or a QuantiGene assay.
  • RCA Rolling Cycle Amplification
  • SDA strand displacement amplification
  • LAMP Loop-Mediated Isothermal Amplification
  • RPA Recombinase Polymerase Amplification
  • the reporter generated in assay methods disclosed herein is a nucleic acid molecule
  • a unique sequence can be incorporated as an identity barcode (ID) that can be decoded by DNA sequencing or other methods.
  • ID an identity barcode
  • a segment of ID comprising N base nucleotides can generate up to 4 N unique identity codes.
  • FIGs.9A-9B is to directly incorporate the ID into the tag of one binder (FIG.9A) or the tags of both binders (FIG.9B).
  • the nucleic acid reporter contains an ID in the first tag.
  • the nucleic acid reporter contains an ID in the second tag.
  • the nucleic acid reporter contains a first ID in the first tag and a second ID in the second tag.
  • FIG.10 shows an indirect ID barcoding approach, where the IDs are on a separate single-strand nucleic acid molecule hybridized on the corresponding tag of the binder ( i.e . the nucleic acid surrogate).
  • surrogate nucleic acids are used, wherein the first tag or a fragment thereof is complementary to the first surrogate or a fragment thereof, and the second tag or a fragment thereof is complementary to the second surrogate or a fragment thereof.
  • FIG.10A only the first binder is IDed indirectly through a first surrogate.
  • the nucleic acid reporter is generated through ligating the first surrogate with the second tag.
  • both the first and the second binders are IDed indirectly.
  • the reporter can also be generated through proximity extension, collaborative hybridization, and other methods known in the art as disclosed above.
  • the annealing of the ID carrying nucleic acid surrogates with its associated binder tags can be done in reagent manufacturing process before the assay. Alternatively, it can be conducted as part of the assay.
  • the nucleic acid reporter contains a first ID in the first surrogate and a second ID in the second surrogate. In some embodiments, the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second surrogate, and contains an ID in the first surrogate or the second surrogate. In some embodiments, the nucleic acid reporter contains a first ID in the first surrogate and the second ID in the second surrogate.
  • the nucleic acid reporters as disclosed herein contain an analyte-specific ID (the “target ID”). Each analyte is assigned with a unique ID.
  • assay methods comprising simultaneously detecting at least two analytes in the sample by simultaneously detecting the unique target IDs associated with each analyte.
  • the analytes are proteins.
  • the analytes include at least one protein and at least one nucleic acid.
  • the nucleic acid can be DNA or RNA.
  • one ID is sufficient to represent the identity of the target analyte or the immunocomplex formed with the analyte.
  • the ID can be detected and quantified by existing multiplexed nucleic acid detection technologies, such as multiplexed qPCR, multiplexed digital PCR or next generating sequencing (NGS).
  • each of the first binder and the second binder is associated with a unique ID, and only the signals generated from reporter containing the IDs of both binders are counted as true signal.
  • This scheme can be used to reduce or eliminate false positive signal generated by cross-reactivity or non-specific binding among different binding pairs. Accordingly, provided herein are assay methods for detecting analyte in a sample by the co-detection of the first and the second target IDs each associated with the first and second binders, respectively.
  • Incorporation of IDs in the nucleic acid reporters in the assay methods provided herein can also be used to detect interactions between molecules, for example, protein-protein interactions. As depicted in FIG.13, the co-detection and quantification of reporters containing IDs of two binders designed for different molecules would indicate the interaction and affinity of these two associated molecules under the assay condition. In some embodiments, the assay methods provided herein detect protein-protein interactions.
  • analyte in a sample, wherein the analyte is a binding pair of two different proteins; wherein the first binder binds one protein, and the second binder binds the other protein of the binding pair; and wherein the binding pair is detected by the co-detection of the first and the second target IDs.
  • the nucleic acid reporters generated in the assay methods provided herein can also include sample-specific “sample IDs.” As depicted in FIGs.l4A-14C, when the assay method provided herein is performed on a particular sample, a sample ID can be introduced during the assay at or before reporter generation step to identify the sample. With such sample ID incorporated into the reporter, reporters from many samples can be pooled together and read by NGS in parallel.
  • the sample ID can be carried on a nucleic acid independent of the binder tags and incorporated into the reporter at the ligation step as shown in FIG.14A. Accordingly, in some embodiments, the nucleic acid reporter formed in each sample contains an ID that is a sample ID, wherein the sample ID is inserted between the first tag or surrogate, and the second tag or surrogate.
  • the nucleic acid reporter is composed of a fragment of the first tag and a fragment of the second tag, and contains a sample ID inserted between the first tag and the second tag. In some embodiments, the nucleic acid reporter is composed of a fragment of the first tag and a fragment of the second surrogate, and contains a sample ID inserted between the first tag and the second surrogate. In some embodiments, the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second tag, and contains a sample ID inserted between the first surrogate and the second tag. In some embodiments, the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second surrogate, and contains a sample ID inserted between the first surrogate and the second surrogate.
  • the sample ID can be incorporated on a nucleic acid surrogate that is hybridized to a tag during an assay step as shown in FIG.14B.
  • the nucleic acid reporter formed in each sample contains sample ID in the first surrogate or the second surrogate.
  • the nucleic acid reporter is composed of a fragment of the first tag and a fragment of the second surrogate, and contains a sample ID in the second surrogate.
  • the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second tag, and contains a sample ID in the first surrogate.
  • the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second surrogate, and contains a sample ID in the first surrogate or the second surrogate.
  • the sample ID can be incorporated by ligation to a nucleic acid tag or its surrogate during an assay step as shown in FIG.14C.
  • the nucleic acid reporter formed in each sample contains sample ID ligated to the first tag or the second tag, or their respective surrogate.
  • the nucleic acid reporter is composed of a fragment of the first tag and a fragment of the second tag, and contains a sample ID ligated to the first tag or the second tag.
  • the nucleic acid reporter is composed of a fragment of the first tag and a fragment of the second surrogate, and contains a sample ID ligated to the first tag or the second surrogate.
  • the assay methods provided herein simultaneously detect an analyte in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting the unique sample IDs associated with each sample.
  • the nucleic acid reporters generated in the assay methods provided herein can comprise both a target ID and a sample ID.
  • the nucleic acid reporter comprises (a) a target ID in the first tag or the first surrogate, or in the second tag or the second surrogate, and (b) a sample ID that is (1) inserted between the first tag or surrogate thereof, and the second tag or surrogate thereof, (2) included in the first surrogate or the second surrogate or (3) ligated to the first tag or surrogate thereof, or the second tag or surrogate thereof.
  • the nucleic acid reporter is composed of a fragment of the first tag and a fragment of the second tag, and contains a target ID and a sample ID.
  • the nucleic acid reporter can contain a target ID in the first tag or the second tag.
  • the nucleic acid reporter can contain a first target ID in the first tag and a second target ID in the second tag.
  • the nucleic acid reporter can contain a sample ID inserted between the first tag or the second tag.
  • the nucleic acid reporter is composed of a fragment of the first tag and a fragment of the second surrogate, and contains a target ID and a sample ID.
  • the nucleic acid reporter can contain a target ID in the first tag or the second surrogate.
  • the nucleic acid reporter can contain a first target ID in the first tag and a second target ID in the second surrogate.
  • the nucleic acid reporter can contain a sample ID inserted between the first tag and the second surrogate.
  • the nucleic acid reporter can also contain a sample ID in the second surrogate.
  • the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second tag, and contains a target ID and a sample ID.
  • the nucleic acid reporter can contain a target ID in the first surrogate or the second tag.
  • the nucleic acid reporter can contain a first target ID in the first surrogate and a second target ID in the second tag.
  • the nucleic acid reporter can contain a sample ID inserted between the first surrogate and the second tag.
  • the nucleic acid reporter can also contain a sample ID in the first surrogate.
  • the nucleic acid reporter is composed of a fragment of the first surrogate and a fragment of the second surrogate, and contains a target ID and a sample ID.
  • the nucleic acid reporter can contain a target ID in the first surrogate or the second surrogate.
  • the nuclei c acid reporter can contain a first target ID in the first surrogate and a second target ID in the second surrogate.
  • the nucleic acid reporter can contain a sample ID inserted between the first surrogate and the second surrogate.
  • the nucleic acid reporter can also contain a sample ID in the first surrogate or the second surrogate.
  • assay methods comprising simultaneously detecting at least two analytes in at least two samples, by simultaneously detecting the unique sample IDs and unique target IDs associated with each analyte and the unique sample IDs associated with each sample.
  • the assay methods provided herein simultaneously detect at least two analytes in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting unique sample IDs and unique target IDs in the nucleic acid reporters with each sample.
  • the assay methods provided herein simultaneously detect at least five analytes in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting unique sample IDs and unique target IDs in the nucleic acid reporters with each sample.
  • the assay methods provided herein simultaneously detect at least ten analytes in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting unique sample IDs and unique target IDs in the nucleic acid reporters with each sample.
  • the assay methods provided herein simultaneously detect at least fifty analytes in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting unique sample IDs and unique target IDs in the nucleic acid reporters with each sample.
  • the assay methods provided herein simultaneously detect at least eighty analytes in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting unique sample IDs and unique target IDs in the nucleic acid reporters with each sample.
  • the assay methods provided herein simultaneously detect at least one hundred analytes in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting unique sample IDs and unique target IDs in the nucleic acid reporters with each sample.
  • nucleic acid-based capture and ID configurations as described above and shown in FIGs.4A to 7C and 9A-14 can be used in the assay methods disclosed herein.
  • the nucleic acid reporters in the multiplexing assay methods disclosed herein can be detected by multiplexed qPCR, multiplexed digital PCR, or NGS.
  • the nucleic acid reporters in the multiplexing assay methods disclosed herein can be detected by NGS.
  • the use of NGS to detect the nucleic acid reporters generated by assay methods disclosed herein include at least the following advantages.
  • This assay methods provided herein additionally address this need and provide related advantages.
  • assay methods wherein the number of reporter molecules generated from a high-concentration target analyte is reduced in a precise and known proportion so that the limited bandwidth of detection can be efficiently assigned to different target analytes.
  • the assay methods disclosed herein capture the immunocomplexes on a solid surface using a receiving group (e.g . a nucleic acid capture probe) before the reporter is generated, which provides a unique opportunity to reduce the signal of highly abundant analytes by selectively capturing only a portion of the immunocomplexes generated therefrom to the surface.
  • the binders can be conjugated with and without their presenting groups (e.g. nucleic acid tags) in a known proportion. If, for example, binders conjugated with a presenting group are mixed with the same binder without the presenting group at 1% concentration, then only 1% immunocomplex can be captured onto the surface and the reporter to target analyte ratio will be 1%.
  • Nonfunctional presenting groups namely, presenting groups that do not bind receiving groups, can also be used. For example, if only 0.1% of first binder is conjugated to a first presenting group (e.g.
  • the reporter to immunocomplex ratio will be 1:1000, effectively reducing the signals generated by this analyte by 1000 fold.
  • An alternative approach for such partial capture can be used, which introduces a known portion of nonfunctional receiving groups (e.g. nucleic acid capture probes).
  • a certain proportion of the first capture probe can be included that does not have the segment complementary to the universal capture probe (FIG.6B) or is not biotinylated (FIG.6C).
  • the same proportion of immunocomplexes cannot be captured on the surface and thus cannot generate nucleic acid reporters for detection.
  • the reporter to immunocomplex ratio will also be 1 : 1000, reducing the signals generated by this analyte by 1000 fold.
  • assay methods that include proportionally reducing the amount of an analyte detected by the assay, by adding a non-functional binder to the solution in step (1), wherein the non-functional binder competes with the first binder for binding to the analyte but is either unconjugated, or conjugated to a presenting group that does not bind the first receiving group.
  • the non-functional binder is unconjugated.
  • the non-functional binder is conjugated to a presenting group that does not bind the first receiving group.
  • the non-functional binder is conjugated to a nucleic acid tag that cannot hybridize with the first probe coupled to the first solid surface.
  • assay methods that include proportionally reducing the amount of an analyte detected by the assay, by adding a non-functional receiver to the solution in step (1), wherein the non-functional receiver competes with the first receiving for binding to the first presenting group but cannot be coupled with the first solid surface.
  • kits that include proportionally reducing the amount of an analyte detected by the assay, by adding a non functional binder to the solution in step (1), wherein the non-functional binder competes with either the first binder or the second binder for binding to the analyte but forms a immunocomplex that cannot be detected.
  • the assay methods provided herein detect the immunocomplex by detecting a detectable marker conjugated to either the first binder or the second binder, and the non-functional binder is either unconjugated to the detectable marker, or is conjugated to a defective detectable marker that cannot produce the signal for detection.
  • the immunocomplex is detected via the nucleic acid reporter, and the non functional binder can be conjugated to a nucleic acid tag that lacks the proper segment for generating the nucleic acid reporter.
  • the non functional binder can be conjugated to a nucleic acid tag that lacks the proper segment for generating the nucleic acid reporter.
  • an assay method for detecting an analyte in a sample comprising:
  • the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;
  • target label can provide identifiers that can be correlated with the particular target to facilitate the detection and identification of a target molecule.
  • each target label can comprises one or more target IDs.
  • the first target label comprises a first target ID.
  • the second target label comprises a second target ID.
  • the first target label comprises a first target ID and the second target label comprises a second target ID.
  • the reporter can be generated based on proximity between the first target label and the second target label. Accordingly, in one embodiment, the reporter comprises the first target ID. In another embodiment, the reporter comprises the second target ID. In yet another embodiment, the reporter comprises the first target ID the second target ID. In a further embodiment, the reporter comprises the first target ID and the second target ID. In one embodiment, the reporter comprises the first target ID and the sample ID. In another embodiment, the reporter comprises the second target ID and the sample ID. In a further embodiment, the reporter comprises the first target ID, the second target ID, and the sample ID.
  • an assay method for detecting an analyte in a sample comprising: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder, and the sample in a solution, wherein:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • the reporter can be generated based on the properties of the immunocomplex that correlates with the specificity of the binding between both binders and the analyte.
  • the reporter is generated based on proximity between the first target label and the second target label.
  • the reporter is generated based on proximity between the first target ID and the second target ID.
  • the reporter is generated based on proximity between the first binder and the second binder.
  • an assay method for detecting an analyte in a sample comprising:
  • the reporter is a non-nucleic acid reporter.
  • the reporter is a nucleic acid reporter.
  • the method provided herein comprises:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • nucleic acid reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first and the second target ID;
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;
  • methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;
  • methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;
  • methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first and the second target ID;
  • methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first and the second target ID;
  • methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID; (2) washing the first solid surface to remove unbound molecules;
  • ID identity barcode
  • target ID analyte-specific
  • reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first and the second target ID;
  • the immunocomplex can be released from the first surface and recaptured in the second surface to further increase the signal to noise ratio.
  • FIGs. 21A-21F depict exemplary schematic representations of an assay method including such two captures.
  • the second binding moiety further comprises a second presenting group.
  • the method further comprises a step 2(b) between step 2(a) and step (3): (2b) introducing a second solid surface and recapturing the immunocomplex on the second solid surface via binding between the second presenting group and the second receiving group coupled to the second solid surface.
  • the methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;
  • the methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first and the second target ID;
  • the methods provided herein further comprise a step 2(c) between step 2(b) and step (3): (2c) washing the second solid surface to remove unbound molecules.
  • the methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;
  • the methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first and the second target ID;
  • the methods provided herein further comprise a step 2(d): releasing the immunocomplex from the second solid surface by disrupting the binding between the second presenting group and the second receiving group.
  • the methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;
  • the methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and (iii) the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID first identity barcode
  • target ID analyte-specific
  • reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first and the second target ID;
  • the methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;
  • the methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • the reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first and the second target ID;
  • the methods provided herein comprise:
  • the first and second binders bind to the analyte and form an immunocomplex
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface
  • the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;
  • the methods provided herein comprise:
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • reporter comprises (i) the first target ID, (ii) the second target ID, or (iii) both the first and the second target ID;
  • an assay method for detecting an analyte in a sample comprising:
  • the first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • FIGs. 20A-20D provide exemplary schematic representations of generating a nucleic acid reporter after being released from a solid surface (indicated as 1st surface in FIGs. 20B-20C) and FIG. 20E provides confirmatory data showing from such an exemplary capture and release assay, in which the analyte is detected by signals determined via the target ID in the target label as generated in the nucleic acid reporter.
  • the method provided herein further comprises a step (2a) between step (2) and (3): releasing the immunocomplex from the first solid surface by disrupting the binding between the first presenting group and the first receiving group.
  • the method provided herein comprises:
  • the first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • the immunocomplex can be released from the first surface and recaptured in the second surface to further increase the signal to noise ratio.
  • FIGs. 21A-21F depict exemplary schematic representations of an assay method including such two captures.
  • the second binding moiety further comprises a second presenting group.
  • the method further comprises a step 2(b) and a step 2(c) between step 2(a) and step (3): (2b) introducing a second solid surface and recapturing the immunocomplex on the second solid surface via binding between the second presenting group and the second receiving group coupled to the second solid surface; and (2c) washing the second solid surface to remove unbound molecules.
  • the methods provided herein comprises:
  • the first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • FIGs. 21G-21I provide exemplary schematic representations of generating a nucleic acid reporter after being captured and released from 1st and 2nd solid surface and FIGs. 24A-24B and 25A-25C provide confirmatory data showing such an exemplary capture and release assay, in which the analyte is detected by signals determined via the target ID in the target label as generated in the nucleic acid reporter.
  • the method provided herein further comprises a step (2d): releasing the immunocomplex from the second solid surface by disrupting the binding between the second presenting group and the second receiving group.
  • the method provided herein comprises: (1) mixing a first binding moiety comprising a first binder and a first presenting group, a second binding moiety comprising a second binder and a second presenting group, and the sample in a solution, wherein:
  • the first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • the nucleic acid reporters generated in the assay methods provided herein can also include sample-specific “sample IDs.”
  • FIGs. 22 and FIGs. 23 A-23B provide exemplary schematic representations for generating a nucleic acid reporter with sample IDs and FIGs. 24A-24B and 25A-25C provide confirmatory data showing such multiplexing assays, wherein the analyte is detected by signals determined via the target ID in the target label and the sample ID as generated in the nucleic acid reporter, thereby parsing out the samples via the sample IDs and analyte via the target IDs.
  • the method provided herein further comprises a step (2e): binding a sample label comprising an ID that is sample-specific (“sample ID”) (i) to the first target label, (ii) to the second target label, or (iii) to both the first target label and the second target label.
  • sample ID an ID that is sample-specific
  • the method provided herein comprises:
  • the first and second binders bind non-interfering epitopes on the analyte and form an immunocomplex
  • the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and
  • the first binding moiety further comprises a first target label comprising a first identity barcode (“ID”) that is analyte-specific (“target ID”) and the second binding moiety further comprises a second target label comprising a second target ID;
  • ID identity barcode
  • target ID analyte-specific
  • sample ID an ID that is sample-specific (“sample ID”) (i) to the first target label, (ii) to the second target label, or (iii) to both the first target label and the second target label
  • Each binding moiety can comprise one or more target labels.
  • the first binding moiety comprises one target label.
  • the first binding moiety comprises two target labels.
  • the first binding moiety comprises three target labels.
  • the first binding moiety comprises four target labels.
  • the first binding moiety comprises five or more target labels.
  • the second binding moiety comprises one target label.
  • the second binding moiety comprises two target labels.
  • the second binding moiety comprises three target labels.
  • the second binding moiety comprises four target labels.
  • the second binding moiety comprises five or more target labels.
  • the target labels between the first binding moiety and the second binding moiety are different.
  • the target labels between the first binding moiety and the second binding moiety are identical.
  • the target labels within the first binding moiety are different.
  • the target labels within the first binding moiety are identical.
  • the target labels within the second binding moiety are different.
  • the target labels within the second binding moiety are identical.
  • the first binding moiety have any target labels as provided in this paragraph and the second binding moiety have any target labels as provided in this paragraph in any combination.
  • the first binding moiety comprises a first target label and the second binding moiety comprises a second target label, wherein the first target label and the second target label are different.
  • the first binding moiety comprises a first target label and the second binding moiety comprises a second target label, wherein the first target label and the second target label are identical.
  • one of the two binding moieties can have no target label.
  • the first binding moiety lacks a target label.
  • the second binding moiety lacks a target label.
  • the first binding moiety comprises no target label.
  • the second binding moiety comprises no target label.
  • the first binding moiety comprises no target label and the second binding moiety comprises one target label.
  • the first binding moiety comprises one target label and the second binding moiety comprises no target label.
  • the first binding moiety comprises no target label and the second binding moiety comprises two, three, four, five, or more target labels.
  • the first binding moiety comprises two, three, four, five, or more target labels and the second binding moiety comprises no target label. In one embodiment, the first binding moiety lacks a target label and the second binding moiety comprises one target label. In another embodiment, the first binding moiety comprises one target label and the second binding moiety lacks a target label. In yet another embodiment, the first binding moiety lacks a target label and the second binding moiety comprises two, three, four, five, or more target labels. In another embodiment, the first binding moiety comprises two, three, four, five, or more target labels and the second binding moiety lacks a target label.
  • one of the two binding moieties can have no presenting group.
  • the first binding moiety lacks a presenting group.
  • the second binding moiety lacks a presenting group.
  • the first binding moiety comprises no presenting group.
  • the second binding moiety comprises no presenting group.
  • each target label can comprises one or more target IDs.
  • the first target label comprises a first target ID.
  • the second target label comprises a second target ID.
  • the target IDs between the first target label and the second target label are different. In certain embodiments, the target IDs between the first target label and the second target label are identical. In one specific embodiment, the first target label comprises a first target ID and the second target label comprises a second target ID, wherein the first target ID and the second target ID are different. In another specific embodiment, the first target label comprises a first target ID and the second target label comprises a second target ID, wherein the first target ID and the second target ID are identical.

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