Gonadotrophs constitute 5% or less of the total number of cells in the anterior pituitary of adult female rats. The need for purified gonadotrophs to conduct research in the regulation of gonadotropin secretion in vitro is obvious. Furthermore, heterogeneity among gonadotrophs has been well documented. Antibodies recognizing the differentiation antigens on the cell surface of gonadotrophs may provide a potential means to define and isolate the subtypes of gonadotrophs. Monoclonal antibodies specific to gonadotrophs or their subtypes will be generated using the hybridoma technique, which permits the use of unpurified antigens to obtain homogeneous antibodies with specificities against a single determinant. Enzymatically dispersed and gonadotroph-enriched anterior pituitary cells prepared by unit gravity sedimentation from 14-day old female rats will be used to immunize Balb/c mice. Spleen cells from these mice will be fused with X63-Ag8.653 non-secreting mouse myelomas using polyethylene glycol (PEG). Hybridomas will be plated at low density in HAT medium. Clones will initially be screened for antibodies to cell surface antigens by an enzyme-linked immunoabsorbent assay (ELISA). Positive hybrids will be subsequently screened for antibodies specific to gonadotrophs or to subtypes of gonadotrophs using indirect immunofluorescence staining techniques. Positive clones will be expanded and subsequently recloned by limit dilution. Large amounts of homogeneous antibodies will be produced both in culture and in ascites by growing tumors in Balb/c mice. Gonadotroph-specific monoclonal antibodies will be immobilized on sheep RBC, plastic culture dishes, and Affi-Gel Cell Sorting Beads (Bio-Rad) by an indirect linkage through a second antibody against mouse immunoglobulin or staphylcoccal protein A. These immunoabsorbent surfaces will be employed to purify gonadotrophs by established affinity cell purification techniques. Antibodies specific to the gonadotroph will also be tested for their ability to interact with membrane receptors for gonadotropn-releasing-hormone (GnRH). Antibodies against GnRH receptors will be screened by both radioligand binding assay and immunoprecipitation. In addition, their GnRH agonistic and antagonistic activities will also be examined in an in vitro cell culture system.
NIH ExPorter
NIH ExPorter