Antipeptide antibodies reveal structural and functional characteristics of rat placental lactogen-II

The purpose of this investigation was to develop specific immunologic probes to rat placental lactogen-II (PL-II) and to use the immunologic probes to further characterize rat PL-II. Five oligopeptides corresponding to different regions of rat PL-II (amino acids 1-13, 56-70, 89-103, 107-118, 150-164) were chemically synthesized by solid phase methods and purified to homogeneity by reverse phase high performance liquid chromatography. The synthetic peptides were coupled to keyhole limpet hemocyanin (KLH) and the peptide-KLH conjugates were used to immunize rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA), electrophoresis and immunoblotting analyses. Each of the antipeptide antisera showed reactivity with the entire rat PL-II protein; however, the extent of the reactivities of each antiserum with rat PL-II was dependent on the conformational state of rat PL-H. Antisera directed to amino acids 56-70 showed the best reactivity toward each of the conformational states of rat PL-II tested. Antibodies generated to the entire rat PL-II protein specifically recognized the 56-70 amino acid sequence but showed limited reactivity with synthetic peptide corresponding to amino acids 1-13, 89-103, 107-118, and 150-164 of rat PL-II. Antisera to amino acids 56-70 of rat PL-II were specific for PLs as demonstrated by their recognition of rat PL-II, mouse PL-II and human PL and by their lack of reactivity with rat pituitary prolactin and growth hormone and with a series of other synthetic peptides to rat PL-II and rat prolactin-like protein-A. The immunorecognition of human PL was restricted to antipeptide antibodies directed to amino acids 56-70 of rat PL-II. The chemically synthesized peptides representing various regions of rat PL-II did not show significant interactions with prolactin receptors, and antisera directed to the peptides failed to interfere with the binding of either rat PL-II or human PL to prolactin receptors. In summary, we have generated a series of immunologic probes for studying the structure of rat PL-II. The sequence comprising amino acids 56-70 of rat PL-II was shown to make up at least part of an epitope for rat PL-II and to be a region of significant structural homology with mouse PL-II and human PL.