Inhibition of binding of ovine placental lactogen to growth hormone and prolactin receptors by monoclonal antibodies

Abstract

From a single cell fusion, five stable hybridomas secreting antiovine placental lactogen (oPL) antibodies were obtained. Three of these secrete immunoglobulin (Ig)G subclass, and the other two secrete IgM class antibodies. Ascites fluids were raised in mice for each clone and were used as the antibody component for the development of solid phase RIA. Three solid-phase RIAs were successfully established using individual IgG subclass monoclonal antibodies, but the IgM class antibodies were ineffective. In all three individual solid-phase RIAs, the binding of [125I]iodo-oPL to the immobilized antibody was inhibited by unlabeled oPL, but not by ovine pituitary PRL (oPRL), ovine GH (oGH), or ovine pituitary extract. Two of the IgG subclass antibodies were able to inhibit the binding of [125I] iodo-oPL to PRL receptors(s) and to GH receptor(s) in rabbit mammary gland and liver, respectively. One of these two IgG subclass antibodies was more effective at inhibiting the binding of oPL to PRL receptor(s) in rabbit mammary gland, whereas the other one is more effective in inhibiting the binding of oPL to GH receptor(s) in rabbit liver. These antibodies, however, could only weakly inhibit the binding of [125I]iodo-oPRL to rabbit mammary gland and were ineffective in inhibiting the binding of [125I]iodo-oGH to rabbit liver. The addition of monoclonal antibodies in both radioreceptor assay (RRA) for PRL (RRA-PRL) and for GH (RRA-GH) did not affect the parallelism of the displacement curve of oPL standard. Our results suggest that oPL might contain two distinct binding sequence(s): one responsible for the binding of oPL to PRL receptor(s) and the other responsible for the binding of oPL to GH receptor(s). These two binding sequences might overlap or be located adjacent to one another. The interaction of monoclonal antibodies with these binding sequences of oPL may block the binding of oPL with PRL and GH receptor(s). Alternatively, our studies suggest that the monoclonal antibodies do not bind to hormone receptor(s)-binding sequence(s) in oPL, but the interaction between oPL and monoclonal antibody might alter the conformational structure of the oPL which will consequently lead to a lower binding of oPL to PRL and GH receptor(s).