Radioimmunoassay for the Antigenic Determinants of Cholera Toxin and Its Components

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Abstract
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. Radiolabeled toxin, B subunit and .alpha. chain preparations retained substantial ability to bind to [rabbit] 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 .times. 10-8 .mu.mol/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 whole toxin or its components. Apparently, 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.