Purification of subunits from human chorionic gonadotropin and radioimmunoassay

Abstract

A crude hCG with an activity of about 3,000 IU per mg was purifed to 10,000--15,000 IU per mg of dry weight using Amberlite CG-50 chromatography combined with DEAE-Sephadex A-50 and Sephadex G-75. The alpha and beta subunits of hCG were prepared by urea-treatment of the hormone and isolated by DEAE-Sephadex A-50 chromatography. Further purification of the subunits was achieved by gel filtration on a Sephadex G-76 column. For radioimmunoassay hCG was iodinated by the DMSO-chloramine T method. Iodination of hCG with non-radioactive iodine revealed that the addition of DMSO to the iodination mixture seemed to reduce the iodination damage to the antigenic activity of the hormone. Non-radioactive iodine substituted hCG accomplished by the DMSO-chloramine T method showed 1.5 times more immunoreactive in the hCG radioimmunoassay than hCG iodinated by the usual chloramine T method. The radioimmunoassay of the hCG-beta subunit developed in our laboratory was satisfactory with respect to specificity; hLH, hFSH, hTSH and hCG-alpha subunit tested were cross-reacted very poorly in our assay system. Desialylated-hCG and subunits, whose biologic potency was almost zero, exhibited also decreased immunoreactivity, about 30% of the native hormones with grossly unimpaired parallelism in their respective homologous radioimmunoassays. The concentrations of hCG and the subunits were determined on human sera from pregnant patients during the course of pregnancy. The hCG levels reached to the peak at the first trimester of the pregnancy, however, the hCG-beta subunits varied in their concentrations poorly throughout the pregnancy periods. The hCG-alpha levels, on the other hand, depicted two distinct peaks, at the early period and the term of pregnancy.