Production and characterization of monoclonal antibodies to purified deglycosylated cystic fibrosis respiratory mucin: evidence for the presence of four immunologically distinct epitopes.
Respiratory mucus glycoproteins (mucins) were purified from the tracheobronchial secretions of three Cystic Fibrosis (CF) patients. The mucins were completely deglycosylated by treatment with trifluoromethanesulfonic acid and subsequent treatment with alpha-N-acetylgalactosaminidase. Over thirty hybrid clones secreting antibodies against the deglycosylated mucin (DGM) were obtained using standard hybridoma techniques. Hybrids with positive identification for CF-DGM were cloned twice using limiting dilution method to ensure the monoclonal nature of the antibodies. Eight stable clones (1a, 1b, 10a, 10c, 10d, 10e, 29d, and 30e) secreting monoclonal antibodies (MAbs) showing specificity of reaction to CF-DGM were obtained. Two clones, 29d and 30e, secreted antibodies of the IgM class while the other six clones secreted antibodies of the IgG1 subclass. Denaturation and reduction experiments suggested that MAbs 1b, 10e, 29d and 30e were directed against a given sequence of amino acids in the DGM while the other four MAbs, in addition to being sequence specific, were also conformation dependent. Further, competitive binding radioimmunoassays suggested that MAbs 1b, 10e, 29d and 30e recognize four distinct epitopes in the peptidic core of CF respiratory mucin. In summary, the MAbs may provide a promising approach to elucidate the structure of the polypeptide backbone of human respiratory mucins as well as for the screening of cDNA libraries for clones secreting mucin(s).
Desai
V C
VC
College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Naziruddin
B
B
Graves
D C
DC
Reyes de la Rocha
S
S
Sachdev
G P
GP
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D001980:Bronchi / Q000276:immunology; D003550:Cystic Fibrosis / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D005260:Female; D006031:Glycosylation; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009077:Mucins / Q000737:chemistry / Q000276:immunology*; D009093:Mucus / Q000276:immunology; D014132:Trachea / Q000276:immunology
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000939:Epitopes; D009077:Mucins