NON-EQUILIBRIUM TWO-SITE ASSAYS FOR LINEAR, ULTRASENSITIVE ANALYTE DETECTION

摘要

Methods and kits related to non-equilibrium, ultrasensitive two-site assays for detecting analytes are provided. In one aspect, a two-site assays for detecting analytes under non-equilibrium analyte binding conditions, using low concentrations of reporter specificity molecule (e.g., reporter antibody) and kits for performing the same is provided. In another aspect, methods for selecting antibodies or specificity molecules with low dissociation constants for use as reporter antibodies in non-equilibrium two-site immunoassays, including two-site immuno-PCR assays, and assays performed with those antibodies, are also provided.

主权项

1. A method for detecting a non-nucleic acid analyte present in a sample to be tested using a forward two-site immuno-PCR assay, the method comprising the steps of:
(1) contacting the sample containing the analyte with:
(a) a reporter conjugate present at a concentration ranging from about 1 to 15 pM or from about 0.15 to 2.25 ng/mL, wherein the reporter conjugate comprises:
(i) a reporter monoclonal antibody capable of specifically binding the analyte in the test sample with a dissociation rate constant lower than about 3.0×10−4 sec−1; and(ii) a nucleic acid marker;thereby forming a first immune complex; and (2) contacting the first immune complex with a capture monoclonal antibody bound to a solid support, where the second antibody is capable of specifically binding to the analyte in the first immune complex, thereby forming a two-site immune complex bound to the support; (3) washing the two-site immune complex bound to the support; and (4) detecting the presence and amount of the nucleic acid marker in the two-site immune complex, wherein the assay detects analyte present in the sample at concentrations of less than 10 picogram/ml.