A specific radio immunoassay of a human chorionic gonadotropin beta subunit in the presence of native human chorionic gonadotropin and immunological biological and chemical characterization of human chorionic gonadotropin and its chemically modified subunits

Okumura, H.; Sudo, T.; Sawai, M.

Folia Endocrinologica Japonica 55(1): 38-53


Accession: 004629516

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A specific radioimmunoassay (RIA) of a hCG-.beta. subunit independent of native hCG by using a specific antiserum of the hCG-.beta. subunit. The antiserum obtained by an absorption procedure was specific enough to assay the hCG-.beta. subunit in serum and urinary specimens collected from pregnant women and a subject with a cancerous disease. A rabbit antibody against the conformational specificity of antigenic determinants of the hCG-.beta. subunit was obtained by the following procedures: A chemically purified and fractionated hCG-.beta. subunit was 1st purified by an absorption technique using anti-normal human serum and anti-urinary Albert's B fraction antisera. The antigen was then absorbed and purified by an anti-hCG antiserum which has a higher association constant (Ka) for antigenic determinant of native hCG and a lower Ka for the hCG-.beta. subunit. The antiserum was obtained by multiple intradermal injections of about 100 .mu.g of the antigen thus purified. The antibody against native hCG in the anti-hCG-.beta. subunit antiserum with higher Ka for the hCG-.beta. subunit was neutralized with highly purified parent hCG. The cross reaction between the anti-hCG-.beta. subunit antisera thus purified and hCG was only from 2.7 to 3.3%, whereas with the anti-hCG-.beta. subunit antisera without the neutralization procedure, there was from 6.3 to 11.0% by Ka calculation or 50% binding in RIA. The peak levels of the HCG-.beta. subunit in the urine were obtained at the 11th-13th weeks after pregnancy, whereas the peak did not appear in the serum specimens in the purified specific RIA. The urine from a patient with gastric carcinoma was characterized by a gradual elevation of the specific hCG-.beta. subunits in contrast with minor changes of antigens determined by the conventional RIA, the values of which were possibly under the influence of native hCG levels. The chemical and enzymatic effects of hCG and its .alpha.- and .beta.-subunits were studied on immunological, biological and chemical behavior. Using citraconic and maleic acids for the modification of hCG and its subunits, Lys residues in the hCG-.alpha. subunit and arginine residues in the hCG-.beta. subunit proved strongly responsible for biological activity. The plasmin digestion of hCG for 3 h gave a strong formation of immune complex with the anti-hCG-.beta. subunit antiserum and for 8 h formed a strong precipitin line with antiserum to the hCG-.alpha. subunit by gel diffusion analysis. These results show further possibilities for developing specific assays for hCG subunits.