Epitope mapping of monoclonal antibodies to bovine prolactin.
The epitopes recognized by three monoclonal antibodies generated to sheep prolactin were determined by evaluating their cross-reactivities by immunodot analysis with 14 mutants of bovine prolactin, in which individual amino acids had been deleted or substituted. Mutations were made throughout the molecule and included disruption of the amino-terminal, carboxyl-terminal, and central disulfide loops. Lack of immunoreactivity was taken as an indication that the site of mutation was part of the epitope. Antibody 6F11 reacted with all bovine prolactin mutants tested, except those in which the carboxyl-terminal cysteine (position 199) was substituted by a serine. Antibodies 5G2 and 4C10 reacted with all of the bovine prolactin mutants, except those in which the amino-terminal cysteine (position 4) was substituted by a serine. Western blot analysis of sheep, squirrel monkey, and rat prolactins with the monoclonal antibodies revealed that 5G2 and 4C10 were specific for sheep prolactin, whereas antibody 6F11 cross-reacted with prolactins from all three species. The mitogenic activity of sheep or rat prolactin in the Nb2 bioassay was determined in the presence of the antibodies to determine whether the epitopes were part of the functional domains of these prolactins. The bioactivity of sheep prolactin (0.4 ng/ml) was unaffected by the monoclonal antibodies [0.01-1 microgram immunoglobulin G (IgG)/ml], whereas the bioactivity of rat prolactin (1.25 ng/ml) was inhibited by 6F11 with an apparent 50% inhibitory concentration of 0.25 microgram IgG/ml. These results indicate that monoclonal antibodies 5G2 and 4C10 cross-react with a species-specific region of the amino-terminal disulfide loop of bovine prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)
Scammell
J G
JG
Department of Pharmacology, University of South Alabama, College of Medicine, Mobile 36688.
Luck
D N
DN
Valentine
D L
DL
Smith
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000502:physiology; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D000939:Epitopes; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D011388:Prolactin / Q000276:immunology* / Q000502:physiology; D051381:Rats; D012453:Saimiri; D012756:Sheep; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015345:Oligonucleotide Probes; D011388:Prolactin