Comparison of in Vivo and in Vitro Neutralization of Human Chorionic Gonadotropin (hCG) Activities by Antisera to hCG and a Carboxy-Terminal Fragment of the β-Subunit*

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Abstract
Unlike antisera to native hCG or its whole α- or β-subunits, antisera to the unique COOH-terminal peptide of the hCG β-subunit or to analogous synthetic peptides fail to neutralize the biological activity of hCG in conventional in vivo bioassays. Whether this difference is due to the dissociation of hCG (free) from the hormone-antibody complex or to the retention of the intrinsic biological activity of hCG even when bound to the antibody is not known. To resolve this question, the effects of antisera on hCG activities were compared in vitro and in vivo, including the metabolic fates of hCG-antibody complexes. In the in vitro system, the addition of increasing amounts of anti-hCGβ-COOH-terminal peptide serum (Abe) obviated [125>I]iodo-hCG binding to rat testis homogenates and inhibited hCG-stimulated testosterone production in the collagenase-dispersed rat interstitial cell assay. Furthermore, the [125I]iodo-Abc, purified by affinity chromatography and in the presence of graded amounts of hCG (0.1 ng to 10 μg), failed to bind in testis homogenates, indicating that the hCG-Abc complex did not bind to the testis receptors. In in vivo experiments, the metabolic fates of [125I]iodo-hCG and [125I]iodo-hCG-Abc were compared with hCG-[125I]iodo-Abc, [125I]iodo-Abc, and [125I]iodo-hCG-antiserum to native hCG (Abh) in intact adult male rats. The plasma half-life of [125I]iodo-hCG-Abc and the uptake of radioactivity into testis, kidney, and liver were between those of [125I]iodo-hCG and hCG-[125I]iodo-Abc, [125I]iodo-Abc, or [125I]- iodo-hCG-Abn. That the metabolic fates of [125I]iodo-hCG-Abc and hCG-[125I]iodo-Abc, as measured by radioactivity, were significantly different, and free hCG was detected in sera injected with the complex suggest that hCG-Abc became dissociated during circulation, and each component was metabolized independently. Although the hCG-Abc did not stimulate testosterone production in intact male rats within 2 h, it was associated with an eventual rise (P < 0.01) in serum testosterone, equivalent to that after hCG injection. These findings indicate a lag time of up to 2 h, but less than 4 h, for significant dissociation of this hormone-antibody complex in vivo, whereafter free hCG stimulated steroidogenesis. Collectively, these observations suggest that the hCG-Abc complex is not active in vitro, and its apparent in vivo biological activity arises from free hCG after its belated dissociation from the antibodies. The apparent association constant of hCG-Abc of 1.2 × 109 M-1, 50 times lower than that of hCG-Abn and 20 times lower than that of hCG-gonadal receptor, supports this interpretation. Further, our findings demonstrate the risks of developing an antifertility vaccine using restricted portions of hCGβ for avoidance of LH cross-reactivity where the hormoneantibody complex may become dissociated in the circulation. Accordingly, in vitro bioassays are not sufficient for assessment of the immune status of women receiving these antigens.