Immunofluorometry of choriogonadotropin by time-resolved fluorescence spectroscopy, with a new europium chelate as label.

Abstract

We describe a new "sandwich"-type non-isotopic immunoassay for human choriogonadotropin (hCG) in serum. In the assay, hCG is captured by a beta-subunit-specific monoclonal antibody, which is immobilized in a white microtiter well. The sandwich is completed by adding a second biotinylated monoclonal antibody specific for the whole hCG molecule. The degree of binding of biotinylated antibody, which is proportional to the amount of hCG present in the sample, is quantified by adding streptavidin labeled with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), in the presence of excess Eu3+. The fluorescent complex formed on the solid-phase [monoclonal antibody-hCG-monoclonal antibody-biotin-streptavidin-BCPDA-Eu3+] is measured by excitation at 337.1 nm with a nitrogen laser and monitoring the emission at 615 nm in a specially designed gated fluorometer working in a time-resolved mode. A two-step procedure is proposed for routine use to avoid the "high-dose hook effect" of the simpler and faster one-step procedure. The hCG assay described has a dynamic range of 1 to 500 int. units/L, and is precise and accurate. Results agree well with those obtained with a commercially available immunoradiometric and a time-resolved immunofluorometric procedure.