Donini S, D'Alessio I, Donini P
Acta Endocrinol (Copenh). 1975 Aug;79(4):749-66. doi: 10.1530/acta.0.0790749.
The alpha and beta subunits of human chorionic gonadotrophin (hCG) were prepared by incubation in 8 M urea, pH 4.5. The separation of the two subunits was obtained by DEAE-Sephadex A-25 chromatography and purification was carried out by gel filtration on Sephadex G-100. The beta subunit obtained was biologically active and was therefore further purified by affinity chromatography using as immuno-adsorbent the alpha antibodies coupled to Sepharose 4B. The beta subunit so purified showed a biological activity less than 1 IU/mg. The immunological and biological properties of the hCG subunits have been studied. It was found that the anti HCG beta serum can discriminate between hCG and hLH and that in the 125I-hCG + anti-beta serum radioimmunoassay, the cross-reactivity of pituitary hLH was lower than that of urinary hLH. Moreover, it was observed that the less purified was the urinary LH preparation, the higher was the cross-reactivity. Therefore we considered the hypothesis that during the purification of human menopausal gonadotrophin (hMG) some LH subunits or smaller immunoreactive fragments could have been discarded with the waste fractions. In order to test the validity of this hypophysis, all the protein fractions obtained during the purification of the hMG were gel-filtered on Sephadex G-100. The immunoreactivity of the effluents from the gel filtration was tested by hCG, hCG-beta, hCG-alpha and hLH radioimmunoassays. While the alpha reactive material was found in some fractions as a peak having the same Ve/Vo value as hCG-alpha, the beta reactive material presenude hMG fractions was not observed in other fractions. The cross-reactivity with the anti beta serum was very low and was found in the LH region of the gel chromatogram. Furthermore, the neutralization of the biological activity of hCG and of urinary and pituitary LH by the anti hCG beta serum was studied by incubating a fixed amount of the three hormones with increasing volumes of antiserum and measuring the LH ACTIVITY AFTER INCUBATION BY THE OADD test. It was observed that the anti hCG beta serum inhibits hCG more than urinary or pituitary LH.
人绒毛膜促性腺激素（hCG）的α和β亚基通过在pH 4.5的8 M尿素中孵育制备。通过DEAE-葡聚糖凝胶A-25色谱法分离这两个亚基，并通过Sephadex G-100凝胶过滤进行纯化。得到的β亚基具有生物活性，因此进一步通过亲和色谱法纯化，使用偶联到琼脂糖4B上的α抗体作为免疫吸附剂。如此纯化的β亚基显示出的生物活性小于1 IU/mg。对hCG亚基的免疫学和生物学特性进行了研究。发现抗HCG β血清可以区分hCG和hLH，并且在125I-hCG +抗β血清放射免疫测定中，垂体hLH的交叉反应性低于尿hLH。此外，观察到尿LH制剂纯化程度越低，交叉反应性越高。因此，我们考虑了这样一种假设，即在人绝经期促性腺激素（hMG）的纯化过程中，一些LH亚基或较小的免疫反应性片段可能已随废弃部分被丢弃。为了检验这一假设的正确性，将hMG纯化过程中获得的所有蛋白质组分在Sephadex G-100上进行凝胶过滤。通过hCG、hCG-β、hCG-α和hLH放射免疫测定来检测凝胶过滤流出物的免疫反应性。虽然在某些组分中发现了α反应性物质，其峰的Ve/Vo值与hCG-α相同，但在其他组分中未观察到存在于hMG组分中的β反应性物质。与抗β血清的交叉反应性非常低，且出现在凝胶色谱图的LH区域。此外，通过将固定量的三种激素与逐渐增加体积的抗血清孵育，并通过OADD试验测量孵育后的LH活性，研究了抗hCG β血清对hCG以及尿和垂体LH生物活性的中和作用。观察到抗hCG β血清对hCG的抑制作用大于对尿或垂体LH的抑制作用。
