Endocrinology, Vol 127, 2090-2098, Copyright © 1990 by Endocrine Society

ARTICLES

Monoclonal antibodies against luteinizing hormone receptor. Immunochemical characterization of the receptor

MT Vuhai-Luuthi, A Jolivet, B Jallal, R Salesse, JM Bidart, A Houllier, A Guiochon-Mantel, J Garnier and E Milgrom
Institut National de la Sante et de la Recherche Medicale, Unite 135, Hormones et Reproduction, Hopital de Bicetre, Le Kremlin-Bicetre, France.

Human CG (hCG)-receptor complexes were solubilized from porcine testicular membranes. They were chromatographed on an immunomatrix of Affi-Gel 10-D1E8 anti beta-hCG monoclonal antibody (this antibody has been shown not to interfere with hCG binding to receptor). Elution was performed at alkaline pH, a condition in which hCG-receptor complexes are relatively stable. Immunization of a mouse with these partially (approximately 15%) purified hormone-receptor complexes allowed the preparation of 20 different hybridomas, each secreting antireceptor antibodies. The latter were used for receptor characterization. Immunoblot of testicular membrane extracts or of purified receptor preparations showed the presence of a major band at approximately 85,000 mol wt and minor bands at approximately 68,000 mol wt and approximately 48-45,000 mol wt. The width of all these bands suggested some microheterogeneity possibly due to glycosylation. The same approximately 85,000 mol wt receptor was seen in ovarian membranes, but no detectable antigen was observed in liver, muscle, and kidney membranes. An immunoaffinity method (using antibody LHR 38) was devised to purify the receptor in a single step. This demonstrated that the purified receptor preparation did not contain any protein component other than those detected by immunoblot. Comparison of receptors purified by immunoaffinity chromatography using either antireceptor or antihormone monoclonal antibodies showed in both cases the presence of the 85,000 mol wt and 48-45,000 mol wt species, but the absence, in the latter case, of the 68,000 mol wt species. This suggests that the 68,000 mol wt receptor cannot bind hormone and does not form oligomers with other receptor species.

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