Enzyme-catalyzed metal deposition for the enhanced detection of analytes of interest
First Claim
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1. A method of detecting and quantifying an analyte of interest from a biological sample in an enzyme immunoassay (EIA), comprising the steps of:
- (a) contacting said biological sample with a primary antibody immobilized on a solid support, such that a primary antibody-analyte complex will form when analyte is present in the biological sample;
(b) contacting said complex formed in step (a) with a secondary antibody conjugated to a label enzyme selected from the group consisting of alkaline phosphatase, acid phosphatase, alpha- and beta-galactosidases, and esterases, such that a primary antibody-analyte-secondary antibody complex will form when analyte is present in the biological sample;
(c) contacting said primary antibody-analyte-secondary antibody complex formed in step (b) with a redox inactive reductive species in the presence of metal ion, wherein said redox inactive reductive species is a substrate for said label enzyme, and wherein said label enzyme converts said redox inactive reductive species to a redox active reductive species that reduces said metal ion such that a metal precipitate will form on the solid support following reduction of said metal ion;
(d) enhancing said metal precipitate; and
(e) detecting and quantifying the analyte in the biological sample by quantifying the metal precipitate formed on the solid support.
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Abstract
The invention is directed to enhanced methods for detecting an analyte of interest in situ, by immunoassay, or by hybridization comprising binding an enzyme-labeled conjugate molecule to an analyte of interest in the presence of a redox-inactive reductive species and a soluble metal ion. The enzyme catalyzes the conversion of the inactive reductive species to an active reducing agent, which in turn reduces the metal ion to a metal atom thereby providing an enhanced means of detecting the analyte via metal deposition.
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14 Claims
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1. A method of detecting and quantifying an analyte of interest from a biological sample in an enzyme immunoassay (EIA), comprising the steps of:
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(a) contacting said biological sample with a primary antibody immobilized on a solid support, such that a primary antibody-analyte complex will form when analyte is present in the biological sample; (b) contacting said complex formed in step (a) with a secondary antibody conjugated to a label enzyme selected from the group consisting of alkaline phosphatase, acid phosphatase, alpha- and beta-galactosidases, and esterases, such that a primary antibody-analyte-secondary antibody complex will form when analyte is present in the biological sample; (c) contacting said primary antibody-analyte-secondary antibody complex formed in step (b) with a redox inactive reductive species in the presence of metal ion, wherein said redox inactive reductive species is a substrate for said label enzyme, and wherein said label enzyme converts said redox inactive reductive species to a redox active reductive species that reduces said metal ion such that a metal precipitate will form on the solid support following reduction of said metal ion; (d) enhancing said metal precipitate; and (e) detecting and quantifying the analyte in the biological sample by quantifying the metal precipitate formed on the solid support. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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