Production of monoclonal antibodies against the N-terminal glycopeptide of porcine pro-opiomelanocortin: their use for solid-phase radioimmunoassay, western blotting, and immunogold cytochemistry.
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 gamma 3-melanotropin (gamma 3-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.
Chevrier
D
D
Département de biochimie, Université de Montréal, Québec, Canada.
Lemieux
E
E
Fyfe
M
M
Labonté
N
N
Zollinger
M
M
Bennett
H P
HP
Crine
P
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D015153:Blotting, Western; D002417:Cattle; D000939:Epitopes / Q000276:immunology*; D006020:Glycopeptides / Q000276:immunology*; D006801:Humans; D007118:Immunoassay; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology*; D010902:Pituitary Gland / Q000737:chemistry; D011333:Pro-Opiomelanocortin / Q000032:analysis / Q000276:immunology*; D011863:Radioimmunoassay; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006020:Glycopeptides; D010446:Peptide Fragments; D011333:Pro-Opiomelanocortin