Immunoassays exhibiting reduced cross-reactivity with hydrophobic drug analyte metabolites
First Claim
1. An immunoassay for assessing the amount of an analyte of interest in a test sample, wherein the analyte is a hydrophobic drug that metabolizes to form one or more cross-reacting metabolites, the immunoassay comprising the steps of:
- (a) contacting the test sample with one or more pretreatment reagents to form a first mixture, wherein the one or more pretreatment reagents lyse any cells and solubilize any analyte and cross-reacting metabolites present in the test sample;
(b) contacting the first mixture with an antibody specific for the analyte to form a second mixture comprising a complex of the antibody with either analyte or metabolite, wherein the amount of antibody with which the first mixture is contacted is from about 0.1% to about 10% of the amount of analyte in the test sample;
(c) washing the second mixture to remove any analyte and any metabolite not complexed with the antibody and to form a third mixture in which the analyte concentration is decreased to near the equilibrium dissociation constant (KD) of the antibody for analyte wherein the analyte and metabolite concentrations are decreased in the third mixture from about 10-fold to about 500-fold as compared to in the test sample;
(d) contacting the third mixture with a specific binding partner of the antibody labeled with a detectable label (“
tracer”
) to form a fourth mixture comprising a complex of the antibody with tracer (“
antibody-tracer complex”
);
(e) washing the fourth mixture to remove any tracer not complexed with antibody; and
(f) detecting the antibody-tracer complex as measure of the amount of analyte present in the test sample, wherein the immunoassay has less than about 10% cross-reactivity with any one or more cross-reacting metabolites present in the sample.
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Abstract
The present disclosure provides among other things immunoassays exhibiting reduced cross-reactivity with analyte metabolites. Additionally, the present disclosure provides diagnostic immunoassays to determine the concentration or level in a test sample of a hydrophobic drug that metabolizes in vivo or in vitro to form cross-reacting metabolites wherein cross-reactivity with such metabolites of the drug analyte is reduced. In particular, the disclosure provides such immunoassays where the hydrophobic drug is an immunosuppressant drug such as cyclosporine A.
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Citations
28 Claims
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1. An immunoassay for assessing the amount of an analyte of interest in a test sample, wherein the analyte is a hydrophobic drug that metabolizes to form one or more cross-reacting metabolites, the immunoassay comprising the steps of:
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(a) contacting the test sample with one or more pretreatment reagents to form a first mixture, wherein the one or more pretreatment reagents lyse any cells and solubilize any analyte and cross-reacting metabolites present in the test sample; (b) contacting the first mixture with an antibody specific for the analyte to form a second mixture comprising a complex of the antibody with either analyte or metabolite, wherein the amount of antibody with which the first mixture is contacted is from about 0.1% to about 10% of the amount of analyte in the test sample; (c) washing the second mixture to remove any analyte and any metabolite not complexed with the antibody and to form a third mixture in which the analyte concentration is decreased to near the equilibrium dissociation constant (KD) of the antibody for analyte wherein the analyte and metabolite concentrations are decreased in the third mixture from about 10-fold to about 500-fold as compared to in the test sample; (d) contacting the third mixture with a specific binding partner of the antibody labeled with a detectable label (“
tracer”
) to form a fourth mixture comprising a complex of the antibody with tracer (“
antibody-tracer complex”
);(e) washing the fourth mixture to remove any tracer not complexed with antibody; and (f) detecting the antibody-tracer complex as measure of the amount of analyte present in the test sample, wherein the immunoassay has less than about 10% cross-reactivity with any one or more cross-reacting metabolites present in the sample. - 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, 27, 28)
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Specification