Production of monoclonal antibodies against the N-terminal glycopeptide of porcine pro-opiomelanocortin: their use for solid-phase radioimmunoassay, western blotting, and immunogold cytochemistry

Abstract

We have prepared two monoclonal antibodies for the N-terminal glycopeptide of pro-opiomelanocortin 1–77 (N-POMC(1–77)) purified from porcine pituitaries. Antibody 1–244 recognizes an epitope located within the γ₃-melanotropin (γ₃-MSH or POMC(51–77)) sequence, whereas antibody 2–197 binds specifically to a determinant in the 1–49 region of N-POMC. These monoclonal antibodies were used to construct a two-site solid-phase radioimmunoassay that can detect as little as 50 pg of N-POMC(1–77). The assay is linear between 0.5 and 5 ng of porcine peptide and recognizes equally well the homologous peptides purified from human and bovine pituitaries. The assay has been used to analyze reversed-phase high pressure liquid chromatography fractions of crude bovine pituitary extracts and detected a peptide with chromatographic properties identical to those of N-POMC(1–77). When used to stain immunoblots of bovine intermediate pituitary extracts, both the 2–197 and 1–244 antibodies could recognize a major peptide comigrating with purified N-POMC(1–77). In addition, antibody 2–197 also detected a peptide with a mobility similar to that of standard N-POMC(1–49). When used in conjunction with a second anti-mouse antibody coupled to colloidal gold particles, antibody 2–197 stained N-POMC immunoreactive material located in granules in thin sections of pituitary.Key words: monoclonal antibodies, γ-melanotropin, pituitary hormones.