Antigen-coupled immunoreagents

Abstract

The present disclosure provides high-performance immunoreagents for use in a variety of immunologic assays and other related techniques. The immunoreagents comprise a primary antibody and a bridging antigen, wherein the bridging antigen is recognized by a detectable secondary antibody with high affinity. Also provided are compositions comprising panels of immunoreagents specific for multiple different target antigens and compositions comprising pairs of primary immunoreagents and their complementary detectable secondary antibodies. The paired primary immunoreagents and secondary antibodies are useful in a variety of immunologic assays, particularly in highly multiplexed assays, where the structure of the bridging antigen is varied in tandem with variation in the detectable secondary antibody, such that a multiplicity of immunoreagents are provided that are capable of simultaneously detecting a multiplicity of target antigens in a single assay. Also provided are kits comprising the immunoreagents, methods of immunologic assay using the immunoreagents of the disclosure, and methods of preparation of the immunoreagents.

1 - 60 . (canceled) 
     
     
         61 . A method for immunologic assay comprising:
 providing a first sample comprising a first target antigen, a second target antigen, and a third target antigen;   reacting the first target antigen with a first immunoreagent, the second target antigen with a second immunoreagent, and the third target antigen with a third immunoreagent, wherein the first immunoreagent comprises a first primary antibody coupled to a first bridging antigen, wherein the first primary antibody is specific for the first target antigen, wherein the second immunoreagent comprises a second primary antibody coupled to a second bridging antigen, wherein the second primary antibody is specific for the second target antigen, and wherein the third immunoreagent comprises a third primary antibody coupled to a third bridging antigen, wherein the third primary antibody is specific for the third target antigen;   reacting the first immunoreagent with a first detectable secondary antibody, the second immunoreagent with a second detectable secondary antibody, and the third immunoreagent with a third detectable secondary antibody, wherein the first detectable secondary antibody is specific for the first bridging antigen with high affinity, the second detectable secondary antibody is specific for the second bridging antigen with high affinity, and the third detectable secondary antibody is specific for the third bridging antigen with high affinity; and   detecting the first detectable secondary antibody that is associated with the first immunoreagent, the second detectable secondary antibody that is associated with the second immunoreagent, and the third detectable secondary antibody that is associated with the third immunoreagent.   
     
     
         62 . The method of  claim 61 , wherein the first target antigen the second target antigen, or the third target antigen is a cellular marker. 
     
     
         63 . The method of  claim 62 , wherein the cellular marker is selected from the group consisting of: 4-1BB, AFP, ALK1, Amyloid A, Amyloid P, Androgen Receptor, Annexin A1, ASMA, BCA225, BCL-1, BCL-2, BCL-6, BerEP4, Beta-Catenin, Beta-HCG, BG-8, BOB-1, CA19-9, CA125, Calcitonin, Caldesmon, Calponin-1, Calretinin, CAM 5.2, CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD31, CD33, CD34, CD38, CD42b, CD43, CD45 LCA, CD45RO, CD56, CD57, CD61, CD68, CD79a, CD99, CD117, CD138, CD163, CDX2, CEA, Chromogranin A, CMV, c-kit, c-MET, c-MYC, Collagen Type IV, Complement 3c (C3c), COX-2, CXCR5, CK1, CK5, CK6, CK7, CK8, CK14, CK18, CK17, CK19, CK20, CK903, CK AE1, CK AE1/AE3, D2-40, Desmin, DOG-1, E-Cadherin, EGFR, EMA, ER, ERCC1, Factor VIII-RA, Factor XIIIa, Fascin, FoxP1, FoxP3, Galectin-3, GATA-3, GCDFP-15, GCET1, GFAP, Glycophorin A, Glypican 3, Granzyme B, HBME-1,  Helicobacter pylori , Hemoglobin A, Hep Par 1, HER2, HHV-8, HMB-45, HSV 1/11, ICOS, IFNgamma, IgA, IgD, IgG, IgM, IL17, IL4, Inhibin, iNOS, Kappa Ig Light Chain, Ki67, LAG-3, Lambda Ig Light Chain, Lysozyme, Mammaglobin A, MART-1/Melan A, Mast Cell Tryptase, MLH1, MOC-31, MPO, MSA, MSH2, MSH6, MUC1, MUC2, MUM1, MyoD1, Myogenin, Myoglobin, Napsin A, Nestin, NSE, Oct-2, OX40, OX40L, p16, p21, p27, p40, p53, p63, p504s, PAX-5, PAX-8, PD-1, PD-L1, PHH3, PIN-4, PLAP, PMS2,  Pneumocystis jiroveci  ( carinii ), PR, PSA, PSAP, RCC, S-100, SMA, SMM, Smoothelin, SOX10, SOX11, Surfactant Apoprotein A, Synaptophysin, TAG 72, TdT, Thrombomodulin, Thyroglobulin, TIA-1, TIM3, TRAcP, TTF-1, Tyrosinase, Uroplakin, VEGFR-2, Villin, Vimentin, and WT-1. 
     
     
         64 . The method of  claim 61 , wherein the first target antigen, the second target antigen, or the third target antigen is an immunoglobulin from a different species. 
     
     
         65 . The method of  claim 61 , wherein the first detectable secondary antibody, the second detectable secondary antibody, or the third detectable secondary antibody comprises a detectable label. 
     
     
         66 . The method of  claim 65 , wherein the detectable label is a fluorophore, an enzyme, an upconverting nanoparticle, a quantum dot, or a detectable hapten. 
     
     
         67 . The method of  claim 66 , wherein the detectable label is a fluorophore. 
     
     
         68 . The method of  claim 66 , wherein the enzyme is a peroxidase, an alkaline phosphatase, or a glucose oxidase. 
     
     
         69 . The method of  claim 68 , wherein the peroxidase is a horseradish peroxidase or a soybean peroxidase. 
     
     
         70 . The method of  claim 61 , wherein the first detectable secondary antibody, the second detectable secondary antibody, or the third detectable secondary antibody is specific for the first bridging antigen, the second bridging antigen, or the third bridging antigen with a dissociation constant of at most 100 nM, at most 30 nM, at most 10 nM, at most 3 nM, at most 1 nM, at most 0.3 nM, at most 0.1 nM, at most 0.03 nM, at most 0.01 nM, or at most 0.003 nM. 
     
     
         71 . The method of  claim 61 , wherein the first target antigen, the second target antigen, or the third target antigen is within a tissue section. 
     
     
         72 . The method of  claim 71 , wherein the detecting step is a fluorescence detection step. 
     
     
         73 . The method of  claim 71 , wherein the detecting step is an enzymatic detection step. 
     
     
         74 . The method of  claim 61 , wherein the first target antigen, the second target antigen, or the third target antigen is in or on a cell. 
     
     
         75 . The method of  claim 74 , wherein the first target antigen, the second target antigen, or the third target antigen is on the surface of the cell. 
     
     
         76 . The method of  claim 74 , wherein the first target antigen, the second target antigen, or the third target antigen is in the cytoplasm of the cell. 
     
     
         77 . The method of  claim 74 , wherein the first target antigen, the second target antigen, or the third target antigen is in the nucleus of the cell. 
     
     
         78 . The method of  claim 74 , wherein the detecting step is a fluorescence detection step. 
     
     
         79 . The method of  claim 78 , further comprising:
 sorting cells that have bound the first detectable secondary antibody, the second detectable secondary antibody, or the third detectable secondary antibody.   
     
     
         80 - 81 . (canceled) 
     
     
         82 . The method of  claim 61 , further comprising:
 detecting at least five target antigens in the sample.   
     
     
         83 - 122 . (canceled)