Immunological Properties of the β-Subunit of HumanChorionic Gonadotropin (hCG). II. Properties of a hCGSpecific Antibody Prepared against a Chemical Analog of the β-Subunit*

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Abstract
Among the several analogs of the β-subunit of hCG (hCGβ) tested for cross-reactivity with antihuman LH (anti-hLH) in a hLH RIA system, two of them, DS5-hCGβ and DS6-hCGβ, showed no detectable cross-reactivity up to a concentration of 10 μg/ml. DS5-hCGβ and DS6-hCGβ were obtained by the controlled and complete reduction and S-carbamidomethylation of hCGβ, respectively. The derivatives, after treatment with an anti-hLH immunoabsorbent, were coupled with keyhole limpet hemocyanin (hem) and injected in rabbits. The antibodies to DS5,-hCGβ-hem and DS4-hCGβ-hem were highly specific for hCG with no significant cross-reactivity with hLH or other polypeptide hormones. While the antibody to DS5,-hCGβ- hem had a high titer of 1:8000 as well as a high affinity for [I25I]hCG, the anti-DS6-hCGβ-hem was a low affinity and low titer antibody. The specificity and the high titer of the antibody o DS5-hCGβ-hem could be maintained by periodic injection of DS5hCGβ-hem or a mixture of both antigens. The RIA using [125I]hCG and anti-DS55DS6-hCGβ-hem was found to be not only highly specific for hCG but also highly sensitive, with a sensitivity of 0.1–1 ng. The anti-DS5DS6-hCGβMiem appears to be directed toward some site(s) in the amino terminal 116- residue peptide rather than the carboxy-terminal 29-residue peptide, since hCG-des-C-terminus inhibited the binding of [125I]hCG, while the carboxy-terminal 37-residue peptide failed to inhibit. The antibody showed the ability to neutralize specifically in vitro the binding of [125I]hCG, but not of [125I]hLH, to ovarian hCG/hLH receptors. Also, the antibody was able to neutralize in vivo the effect of hCG on ovarian ascorbic acid depletion, while it had no detectable effect o the depletion caused by hLH. Similarly, the anti-DS5/DS6-hCGβ-hem inhibited in vivo the stimulation of the increase in rat uterine weight by hCG, but not that by hLH.