Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain.
Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of 125I-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for 125I-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. 125I-M110 and 125I-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL (ID50 = 0.44 nM) was comparable to that of 125I-oPRL by unlabeled oPRL (ID50 = 0.35 nM), while 125I-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82. These findings indicate that monoclonal antibodies can be readily prepared from partially purified PRL receptors from rabbit mammary gland; two antibodies (M110 and A82) are hormone binding site specific while the other (A917) binds a domain partially but not entirely distinct from the hormone binding site, and that all three antibodies have strong species specificity.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D005260:Female; D007457:Iodine Radioisotopes; D008321:Mammary Glands, Animal / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D011388:Prolactin / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011956:Receptors, Cell Surface / Q000276:immunology* / Q000378:metabolism; D011981:Receptors, Prolactin; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D011981:Receptors, Prolactin; D011388:Prolactin