Positive step immunoassay

Abstract

An assay procedure that is particularly valuable for detecting and/or determining the presence of threshold levels of analyte ligands in biological fluids. In one particularized and specialized aspect the disclosure is directed to procedures for detecting and/or determining threshold levels of hormone metabolites such as pregnanediol-3-glucuronide (P.sub.3 G) and estrone-3-glucuronide (E.sub.1 3G) in human urine. The assay consists of contacting a sample containing the analyte with a known amount of an antibody thereto and with a calibrated amount of the analyte itself that is conjugated to said solid support. When the level of the analyte in the sample exceeds a threshold level, such as 5 ug/ml for P.sub.3 G, the antibody will be insufficient to block all of the corresponding analyte on the solid support. Thus, upon addition of labelled antibody to the assay system, a detectable immunoreaction product becomes attached to the support to indicate that the amount of analyte in the sample exceeds the threshold level. On the other hand, if the level of the analyte in the sample is below the threshold amount, the free antibody will be sufficient to block all of the corresponding analyte on the solid support preventing labelled antibody from forming a detectable immunoreaction product on the support and thus no signal will appear.