title: Monoclonal antibodies to the rabbit liver growth hormone receptor: production and characterization.
abstract: Monoclonal antibodies to the GH receptor (GHR) have been produced by the application of hybridoma technology to splenic lymphocytes from BALB/C mice immunized with a human (hGH) affinity purified preparation of rabbit liver GHR. Primary screening of 384 wells yielded 4 antibodies able to immunoprecipitate [125I]iodo-hGH complexes with purified GHR and one able to inhibit binding of [125I]iodoovine GH ([125I]iodo-oGH) to rabbit liver microsomes. These cells were cloned and grown as ascitic tumors with loss of 1 of the 4 precipitators. Ascitic fluids contained monoclonal antibodies of high titer (inhibitor and 2 precipitators, 1:2.0 X 10(5); one precipitator, 1:2.0 X 10(4)) and high affinity (precipitators, 2.5-6.0 X 10(9) M-1; inhibitor, high affinity component, 6.4 X 10(10) M-1), which were isotyped as IgG1K and IgG2aK (1 precipitator). These antibodies did not cross-react with rabbit insulin or PRL receptors in the appropriate receptor assays and did not possess antihormone activity. Binding of [125I]iodo-MAb7, the inhibitory antibody, was totally blocked by the addition of excess unlabeled oGH or hGH, although these hormones had no effect on binding of the 125I-labeled precipitators. Scatchard analysis of [125I]iodo-oGH binding in the presence of MAb7 showed decreased binding by loss of sites rather than affinity. Antibody dilution curves and Scatchard plots for MAb7 binding provided evidence for two types of GHR in the rabbit liver, in accord with previously published data based on hormone binding studies. All precipitating antibodies gave an enhancement of [125I] iodo-oGH binding with purified receptor (up to 360% of polyethylene glycol-precipitated control), but only minimal enhancement with solubilized microsomal membranes. This enhancement was shown to be due to an increase in receptor number rather than affinity. After examining a number of hypotheses, we concluded that the enhancement was an artifact resulting from a nonpolyethylene glycol-precipitable species of GHR which could be totally precipitated by the monoclonal antibodies. We have produced and characterized four monoclonal antibodies to the GHR which will be of value in characterizing the structure and function of this receptor.
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000939:Epitopes / Q000032:analysis; D013006:Growth Hormone / Q000378:metabolism; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008862:Microsomes, Liver / Q000378:metabolism*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D011986:Receptors, Somatotropin
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D011956:Receptors, Cell Surface; D011986:Receptors, Somatotropin; D013006:Growth Hormone