Detergent-facilitated immunoassay for the rapid and quantitative assay of pharmacological agents

An immunoassay for determining the presence of a target analyte in a test sample comprising the steps of:

• (1) forming a reaction mixture by contacting a test sample with an anti-target analyte antibody capable of binding to a target analyte, a bidentate reagent, and a detergent, wherein the detergent is present in the reaction mixture at a selected concentration greater than about 1%, and wherein the soluble bidentate reagent comprises a biotin member, an analyte member, and a spacer member between the biotin and analyte members, wherein:
• (i) the biotin member of the reagent is capable of binding to a biotin-binding agent selected from the group consisting of avidin and streptavidin, the biotin-binding agent being immobilized to a solid support;
• (ii) the analyte member of the bidentate reagent is capable of specifically binding to the anti-target analyte antibody;
• (iii) the intermediate spacer member is of sufficient length to permit the analyte member to bind to the anti-target analyte antibody and the biotin member to bind to the biotin-binding agent simultaneously;
• and
• (iv) wherein the bidentate reagent is attached to the biotin-binding agent;
• (2) incubating the reaction mixture under conditions sufficient to permit the formation of a complex between the bidentate reagent and the anti-target analyte antibody;
• and
• (3) determining the presence of the target analyte in the test sample by measuring the extent of any formation of the complex, the extent being inversely proportional to the concentration of the target analyte in the sample, and wherein the selected concentration of detergent is determined by the steps of:
• (a) identifying a first, non-zero physiologically relevant, calibrator concentration of the target analyte;
• (b) identifying a second, physiologically relevant, calibrator concentration of the target analyte, wherein the second calibrator concentration is higher than the first calibrator concentration;
• (c) performing steps (1), (2), and (3) at different test detergent concentrations until there is achieved an immunoassay having:
• i) a rate of complex formation greater than about 0.2 O.D. units per minute at a concentration of the target analyte of 0 .mu.g/ml;
• ii) a rate of complex formation greater than about 0.02 O.D. units per minute at the second calibrator concentration;
• iii) a rate of complex formation at the first calibrator concentration equal to at least about 75% of the reaction rate at a concentration of the target analyte of 0 .mu.g/ml;
• and
• iv) a rate of complex formation at the second calibrator concentration of less than 25% of the reaction rate at a concentration of the target analyte of 0 .mu.g/ml.

A homogeneous immunoassay for determining the presence of a target analyte in a test sample comprising the steps of:

• (1) forming a reaction mixture by contacting a test sample with an anti-target analyte antibody capable of binding to a target analyte, a bidentate reagent, a bidentate-binding agent capable of binding to the bidentate reagent, the bidentate-binding agent being immobilized on a latex particle, and a detergent, wherein the detergent is present in the reaction mixture at a selected concentration greater than about 1%, and wherein the bidentate reagent comprises a biotin member, an analyte member, and a spacer member, wherein:
• (i) the biotin member of the reagent is capable of binding to the biotin-binding agent,
• (ii) the analyte member of the bidentate reagent is capable of specifically binding to the anti-target analyte antibody;
• and
• (iii) the intermediate spacer member is of sufficient length to permit the analyte member to bind to the anti-target analyte antibody and the biotin member to bind to the biotin-binding agent simultaneously;
• (2) incubating the reaction mixture under conditions sufficient to permit the formation of a complex of the bidentate reagent, the bidentate-binding agent immobilized on a latex particle, and the anti-target analyte antibody;
• and
• (3) determining the presence of the target analyte in the test sample by measuring the extent of any formation of the complex, the extent being inversely proportional to the concentration of the target analyte in the sample, wherein the selected concentration of detergent to use in the homogeneous immunoassay is determined by the steps of:
• (a) identifying a first, non-zero physiologically relevant, calibrator concentration of the target analyte;
• (b) identifying a second, physiologically relevant, calibrator concentration of the target analyte, wherein the second calibrator concentration is higher than the first calibrator concentration;
• (c) performing steps (1), (2), and (3) at at least two different test detergent concentrations until there is achieved an immunoassay having:
• i) a rate of complex formation greater than about 0.2 O.D. units per minute at a concentration of the target analyte of 0 .mu.g/ml;
• ii) a rate of complex formation greater than about 0.02 O.D. units per minute at the second calibrator concentration;
• iii) a rate of complex formation at the first calibrator concentration equal to at least about 75% of the reaction rate at a concentration of the target analyte of 0 .mu.g/ml;
• and
• iv) a rate complex formation at the second calibrator concentration of less than 25% of the reaction rate at a concentration of the target analyte of 0 .mu.g/ml,
• wherein the detergent is selected from the group consisting of alkylpolyoxyethylene ether, alkylphenylpolyoxyethylene ether and acylpolyoxyethylene sorbitan ester, and
• wherein the latex particle has a diameter from about 38 nanometers to about 100 nanometers.