One-step determination of intact human chorionic gonadotropin by enzyme immuno assay using monoclonal antibodies단일클론 항체를 이용한 인체 융모성 성선 자극 호르몬의 효소 면역학적 측정
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).