Method for carrying out non-isotopic immunoassays, labeled analytes and kits for use in such assays
First Claim
1. A non-isotopic competitive binding assay method for determining the amount of an analyte in a sample containing a known analyte in an unknown concentration, which comprises the steps of:
- (a) forming a reaction mixture by bringing together in a medium, (1) said sample, (2) polypeptide-labeled analyte, (3) an antibody specific for said analyte, (4) a polypeptide partner capable of non-covalently binding with the polypeptides of the polypeptide-labeled analyte to form a complex having catalytic activity, and (5) a substrate capable of being converted to a reporter molecule by the catalytic activity of said complex, said polypeptide-labeled analyte being capable of competitively binding to said antibody and to said polypeptide partner, said antibody inhibiting the formation of a catalytically active complex in the absence of analyte, the concentrations of said antibody, polypeptide partner and polypeptide-labeled analyte being such as to cause varying amounts of analyte in the sample to be directly related to the conversion of said substrate to said reporter molecule as a function of the catalytic activity of the complex;
(b) measuring the conversion of said substrate to said reporter molecule in said reaction mixture, and(c) determining the amount of analyte in said sample by comparing the conversion of said substrate to said reporter molecule to conversions of said substrate to reporter molecule obtained with known concentrations of said analyte.
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Abstract
A highly sensitive, immunoassay method for determining the amount of an analyte in a sample containing a known analyte in an unknown concentration is provided. Sample; a polypeptide-labeled analog of the analyte, an antibody specific for said analyte, a polypeptide partner capable of non-covalently binding with the polypeptide-labeled analyte to form a complex having catalytic activity, and a substrate capable of being converted to a reporter molecule by the catalytic activity of said complex are brought together in a medium. The polypeptide-labeled analyte analog is capable of competitively binding to the antibody and the polypeptide partner, the antibody inhibiting the formation of a catalytically active complex in the absence of analyte, and the concentrations of the antibody, polypeptide partner and polypeptide-labeled analyte are such as to cause varying amounts of analyte to be directly related to the conversion of the substrate to the reporter molecule. Conversion of the substrate to the reporter molecule is then determined, and compared to conversions of substrate to reporter molecule obtained with known concentrations of the analyte.
102 Citations
34 Claims
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1. A non-isotopic competitive binding assay method for determining the amount of an analyte in a sample containing a known analyte in an unknown concentration, which comprises the steps of:
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(a) forming a reaction mixture by bringing together in a medium, (1) said sample, (2) polypeptide-labeled analyte, (3) an antibody specific for said analyte, (4) a polypeptide partner capable of non-covalently binding with the polypeptides of the polypeptide-labeled analyte to form a complex having catalytic activity, and (5) a substrate capable of being converted to a reporter molecule by the catalytic activity of said complex, said polypeptide-labeled analyte being capable of competitively binding to said antibody and to said polypeptide partner, said antibody inhibiting the formation of a catalytically active complex in the absence of analyte, the concentrations of said antibody, polypeptide partner and polypeptide-labeled analyte being such as to cause varying amounts of analyte in the sample to be directly related to the conversion of said substrate to said reporter molecule as a function of the catalytic activity of the complex; (b) measuring the conversion of said substrate to said reporter molecule in said reaction mixture, and (c) determining the amount of analyte in said sample by comparing the conversion of said substrate to said reporter molecule to conversions of said substrate to reporter molecule obtained with known concentrations of said analyte. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A kit for use in carrying out an assay of an analyte, which comprises the following components:
- (1) a polypeptide-labeled analyte, (2) an antibody specific for the analyte, (3) a polypeptide partner capable of non-covalently binding with the polypeptide of the polypeptide-labeled analyte to form a complex having catalytic activity and (4) a substrate capable of being converted to a reporter molecule by the catalytic activity of the complex wherein the polypeptide-labeled analyte, antibody, polypeptide partner and substrate are present in relative amounts sufficient for the determination of the analyte.
- View Dependent Claims (28, 29, 30, 31, 32, 33)
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34. A non-isotopic competitive binding assay method for carrying out a heterogeneous immunoassay of a sample containing a known analyte present in an unknown concentration which comprises:
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(a) forming a reaction mixture by bringing together in a medium (1) said sample, (2) a polypeptide-labeled analyte and (3) an antibody specific for said analyte; (b) separating the antibody-bound, polypeptide-labeled analyte from the free polypeptide-labeled analyte; (c) determining the amount of at least one of;
(1) the antibody-bound, polypeptide-labeled analyte and (2) the free polypeptide-labeled analyte by adding a polypeptide partner capable of non-covalently binding with the polypeptide of the polypeptide-labeled analyte to form a complex having catalytic activity and a substrate capable of being converted to a reporter molecule by the catalytic activity of said complex, by measuring the conversion of said substrate to reporter molecule, and(d) determining the amount of analyte in said sample by comparing the conversion of said substrate to reporter molecule to conversions of said substrate to reporter molecule obtained with known concentrations of said analyte.
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Specification