Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits

Abstract

Hybridoma cell lines which produced monoclonal antibodies against cholera toxin were isolated. These cell lines were detected with an enzyme-linked immunosorbent assay screening procedure with purified cholera toxin or subunit A of cholera toxin. Seven cell lines were characterized with respect to their reactivity with cholera toxin subunits by Western blot analysis. Five clones produced antibodies which were directed against subunit A, and two clones produced antibodies which reacted with subunit B. These antibodies were also characterized by Western blot analysis for reactivity with the heat-labile enterotoxin produced by porcine and human enterotoxinogenic strains of Escherichia coli. Monoclonal antibodies which reacted with subunit A of cholera toxin also reacted with subunit A of both porcine and human heat-labile enterotoxins. In contrast, monoclonal antibodies to subunit B of cholera toxin did not react with subunit B of the heat-labile enterotoxin. Antibodies directed against subunit B neutralized the biological activity of cholera toxin in vitro in the S49 mouse lymphosarcoma assay. In contrast to polyclonal anti-subunit A antisera, monoclonal anti-subunit A from four of five clones had small but measurable neutralizing capacities in vitro.

Characterization of monoclonal antibodies that react with unique and cross-reacting determinants of cholera enterotoxin and its subunits

“…These findings provided additional support for the conclusions that the unique determinants of CT are immunodominant and that CT-B is more immunogenic than CT-A. The isolation of several IgM monoclonal antibodies by Remmers et al (31) and the greater prevalence of monoclonal antibodies specific for CT-A in the report of Robb et al (32) most likely reflected differences in their regimens of immunization.…”

“…We conclude that both unique and cross-reacting determinants on CT-B can elicit neutralizing antibody responses. Monoclonal antibodies against CT-A often have little or no neutralizing activity (24,32). The most potent of three neutralizing monoclonal antibodies against CT-A in our collection (clone 33D2; Table 1) had only 8% of the neutralizing activity of our most potent monoclonal antibody against CT-B (clone 4E2).…”

“…Remmers et al (31) isolated two hybridoma cultures that produced antibodies which reacted both with CT-A and CT-B in solid-phase radioimmunoassays, but the specificity of the crossreactions observed in the radioimmunoassays was not confirmed by an independent method such as the Western blot technique. Robb et al (32) identified a presumptive hybridoma clone that produced monoclonal antibodies against CT-B and CT-A, but additional subcloning demonstrated that the initial clone was impure. No monoclonal antibodies that reacted both with CT-A and CT-B were detected by Lindholm et al (24) or in our study.…”

“…The mixtures were subjected to electrophoresis on vertical 13% polyacrylamide slab gels containing 0.1% SDS by the method of Laemmli (23). Electrophoretic transfer of the proteins from the SDS-polyacrylamide gels to nitrocellulose paper (Schleicher & Schuell Co., Keene, N.H.) and immunological detection of the transferred proteins were performed as described by Robb et al (32). The peroxidase-conjugated rabbit anti-mouse IgG serum used for these experiments was purchased from Miles Laboratories, Inc., Elkhart, Ind.…”

“…Although monoclonal antibodies against CT have been isolated in several laboratories (24,31,32), the number of recognized specificities remains small, and detailed characterization of the epitopes has not yet been reported. In our studies, a variety of screening tests was used to increase the number of different antibody specificities that could be recognized and to extend significantly the results of previous studies of monoclonal anti-CT antibodies.…”