Radioimmunoassay for the antigenic determinants of cholera toxin and its components

A radioimmunoassay procedure is described for the detection of cholera toxin and its component polypeptide chains. Cholera toxin, A subunit, B subunit, alpha chain, and gamma chain were iodinated by the chloramine T procedure. Radiolabeling did not significantly alter the polyacrylamide electrophoretic migration patterns of the toxin or its components. Moreover, radiolabeled toxin, B subunit, and alpha chain preparations retained substantial ability to bind to intestinal mucosal homogenates. The minimal amount of antitoxin detectable with radiolabeled toxin was 0.04 antitoxin units/ml. Substitution of radiolabeled B subunit, A subunit, and alpha chain for radiolabeled toxin decreased the sensitivity of the test. Radiolabeled gamma chain did not bind to the antitoxin preparation. Competitive inhibition studies, with titrated anti-choleragen serum and radiolabeled toxin or components, indicated that the minimum concentration of toxin detectable was 7.0 x 10(-8) mumol/ml at a 90% inhibition level. The A subunit and alpha chain preparations inhibited the binding of the radiolabeled B subunit to antitoxin sites. Conversely, B subunit inhibited the binding of radiolabeled A subunit and alpha chain to antitoxin. The gamma chain did not show any reaction with antitoxin or cross-reaction with either whole toxin or its components. These results strongly suggest that the A subunit and the alpha chain contain antigenic determinant(s) that are common to the B subunit. The B subunit (beta chain) and the alpha chain of cholera toxin may therefore contain region(s) of chemical similarity.