Two polyclonal antisera to rat luteal LH/CG receptor with different ligand binding inhibition and immunohistochemical receptor detection capabilities.
Polyclonal antisera to a SDS-denatured and partially renatured rat luteal 90 K LH/CG receptor were raised in rabbits, characterized, and their applicability for immunohistochemical location of the receptor examined. The LH/CG receptor was purified by hCG-affinity chromatography and subjected either to a preparative SDS-PAGE or Western blotting. Gel slices containing the SDS-denatured or nitrocellulose strips containing the renatured 90 K LH/CG receptor were used for immunization. The antisera, termed ARS-2 and ARS-3, respectively, possessed similar antibody titres. Both antisera were able to recognize the native, SDS-denatured, and SDS-denatured and reduced forms of the LH/CG receptor on dot blots, but only ARS-3 contained antibodies to the hormone binding site or a region near to it, as it was able to inhibit the hCG binding to the membrane-bound LH/CG receptor in a dilution-dependent manner. Both antisera recognized the receptor-hCG complex, but ARS-2 stained the complex with about 50% less intensity than the free receptor. ARS-3 located the LH/CG receptor distinctly on the luteal cell surfaces in immunohistochemical staining with peroxidase antiperoxidase complex method, but ARS-2, although it possessed similar antibody titre, revealed negligible staining. Thus, the antisera readily recognize the native receptor, but differ in their capability for inhibiting hormone binding. Only ARS-3, produced against the renatured receptor, contains sufficient amounts of antibodies capable of recognizing free and occupied receptors in immunohistochemistry.
Lakkakorpi
J T
JT
Biocenter, University of Oulu, Finland.
Keinänen
K P
KP
Rajaniemi
H J
HJ
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007106:Immune Sera / Q000276:immunology*; D007150:Immunohistochemistry; D051381:Rats; D011919:Rats, Inbred Strains; D011967:Receptors, Gonadotropin / Q000276:immunology* / Q000378:metabolism; D011974:Receptors, LH / Q000276:immunology* / Q000378:metabolism
D007106:Immune Sera; D011967:Receptors, Gonadotropin; D011974:Receptors, LH