One-step determination of intact human chorionic gonadotropin by enzyme immuno assay using monoclonal antibodies

Abstract

Human chorionic gonadotropin(hCG) was further purified from crude hCG by Gel filtration (Sephadex G-150). $\alpha$ -and $\beta$ -subunit were separated by treatign 8M Urea and purified by using an ion exchange chromatography (DEAE-Sephadex A-25) and gel filtration (Sephadex G-75). A two-site sandwich EIA for human chorionic gonadotropin employing monoclonal antiboides directed against $\alpha$ -and $\beta$-subunits is described. Monoclonal anti -hCG antibody adn polyclonal rabbit anti $\beta$ -hCG antibody were used for coating mocrotitration plates and monoclonal anti $\beta$ -hCG antibody labelled with alkaline phosphatase was used as a tracer. Monoclonal anti $\alpha$ -hCG, anti $\beta$ -hCG antibody, and rabbit anti $\beta$ -hCG antibody were purified by using whole hCG-affinity-chromatography. The purified Monoclonal antibodies were tested for reactivity with $\alpha-,\beta$-anvd whole hCG by indirect ELISA. The monoclonal anti $\beta$ -hCG antibody strongly reacted with $\beta$ - and whole hCG, and had low reactivity with the $\alpha$ -subunit of hCG. The monoclonal anti $\alpha$ -hCG antibody reacted specifically with $\alpha$ -and whole hCG but hardly with $\beta$ -hCG. Using the two wite EIA, the rabbit anti $\alpha$ -hCG antibody reacted with whole hCG but not with subunits of hCG A two site EIA is able to differentially detect hCG up to 5mIU/ml (1.7ng/ml) with a ``two-step`` assay. The assay can be performed as a ``tow-step`` assay or reduced to a ``one step`` procedure with coincubation of a sample and conjugate. A two site EIA is able to perform the differential detection of whole-hCG in the presence of its subunits and other glycotropic hormones (hTSH, hFSH). This assay system showed some crossreactivity with hLH (7.8\% for a ``two-step`` assay and 13\% for a ``one-step`` assay).