Production and Characterization of Monoclonal Antibodies to Purified Deglycosylated Cystic Fibrosis Respiratory Mucin: Evidence for the Presence of Four Immunologically Distinct Epitopes

Abstract

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).

A novel human airway mucin cDNA encodes a protein with unique tandem-repeat organization

“…Antisera were collected, and the titre was measured against deglycosylated mucin using a standard e.l.i.s.a. [14]. The IgG fraction of the polyclonal antiserum was purified on a Protein A-Superose column (FPLC; Pharmacia).…”

Macromolecular properties and polymeric structure of canine tracheal mucins

“…Purified mucin solubilized (7 mg/ml) in 50 mM-sodium acetate buffer, pH 5.5, containing I mM-CaCl2 was treated with a predetermined (based on mmol of sialic acid present in the native mucins) quantity of neuraminidase ( Vibrio cholerae; Calbiochem) for 24 h at 37°C, dialysed exhaustively against deionized water at 4°C and freeze-dried. Further deglycosylation of the desialylated mucin was then performed by a slight modification of the method of Woodward et al (1987) as described by Desai et al (1991).…”

Monoclonal Antibodies Recognising Sialyl‐Tn: Production and Application to Immunochemistry

“…Co., MO, USA), bovine serum albumen (BSA) (Sigma Chemical Co., MO, USA), and human transferrin (Boehringer-Mannheim, Germany). Native and TFMSA-treated cystic fibrosis (CF) mucin was kindly donated by Dr. Goverdan Sachdev (Desai et al, 1991) while native and TFMSA-treated asthma mucin was kindly provided by Dr. David Thornton (Thornton et al, 1994). Asialomucins were prepared by incubation of 2 mg/ml mucin in PBS with an equal volume of 1 U/ml Vibrio cholerae neuraminidase (Hoechst-Behring, Marburg, Germany) for 3 hours at 3rc.…”