Biological and Immunological Characterization of Crude Commercial Human Choriogonadotropin*

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Abstract
We investigated a commercial hCG preparation to identify and partially characterize the molecular contaminants exhibiting immunological determinants similar to those of hCG itself or biological activities similar to those of the pituitary hormones FSH and TSH. The crude hCG was chromatographed on Sephadex G-100, and the fractions were analyzed for activity in the following assays in which purified hCG is active: rat testis LH/CG radioreceptor assay (RRA), hCGα RIA to a conformational determinant, hCGβ RIA to a conformational determinant, hCGβ carboxy-terminal peptide (CTP) RIA, mouse thyroid bioassay, and ovarian weight bioassay. A single peak of activity in the LH/CG RRA, indicative of intact hCG, coeluted with the peak of hCGyS CTP immunoreactivity. As expected, hCGβ conformational determinant immunoreactivity was found in the same position as hCG, but, surprisingly, its peak level was found in fractions corresponding to the elution position of hCGβ indeed, when these fractions were incubated with purified free hCG α-subunit, recombinant hCG was formed, as judged from an increase in activity in the LH/CG RRA. Thus, in the gel chromatogram, the highest peak of hCGβ conformational immunoreactivity was attributable to hCβ, not to intact hCG. The hCGa immunoreactivity eluted in two major peaks: one corresponding to the position of the intact hCG molecule and the other to a position well after the hCGβ marker. When those fractions containing the hCGa immunoreactivity of smaller apparent molecular size were incubated with purified free hCG β-subunit, no intact hCG with activity in the LH/CG RRA was formed. The follicle-stimulating and thyroid-stimulating activities of the crude hCG preparation eluted in the same position as hCG. Neither of these activities was apparent in fractions where the pituitary hormones FSH and TSH would be expected to elute. The crude hCG preparation contained less than 0.2% O-serine-desialylated hCG by weight, as measured in a RIA directed toward the hCGβ asialo-CTP. Therefore, over 99.8% of the hCG molecules appeared to contain sialic acid in the hCGβ CTP region. These data indicate that 1) the molecule in the crude commercial hCG with activity in biological assays for LH, FSH, and TSH is the intact hCG itself; 2) both free hCGβ3, which can recombine with hCGa, and a form of hCGa which cannot recombine with hCGβ are major immunological contaminantsof the crude hCG; and 3) while heterogeneity of sialic acid content is known to be a property of the hCG in crude preparations, only a small fraction of the hCG exits as a completely desialylated form. (JClin Endocrinol Metab54: 1002, 1982)

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