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The completed sequence of the beta-chain of cholera toxin (103 amino acid residues) was compared to the beta-chains of chorionic gonadotropin, thyrotropin, luteinizing, and follicle stimulating hormones. The overall chemical similarity of the toxin beta-chain to the hormones was not statistically different from random; however, a comparison of the first 40 residues of the toxin beta-chain to the glycoprotein hormones revealed a segment of the hormones which was significantly chemically similar. The probability was less than .003 that the similarity was due to chance.

A radioimmunoassay procedure is described for the detection of cholera toxin and its component polypeptide chains. Cholera toxin, A subunit, B subunit, a chain, and y 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 a 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 a chain for radiolabeled toxin decreased the sensitivity of the test. Radiolabeled y 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 the reaction between antitoxin with the toxin and/or its component polypeptide chains. Preliminary reports of immunological studies assumed the A and B subunits were antigenically 621 on July 31, 2020 by guest http://iai.asm.org/ Downloaded from

Selected rabbit antisera to cholera toxin antigens and convalescent cholera patient sera were analyzed using the permeability factor neutralization test and two sensitive in vitro serological assays specific for cholera toxin, cholera toxin A subunit, and cholera toxin B subunit. The results indicated that antisera to cholera toxin contained toxin-neutralizing activity as well as antibodies specific for both the A subunit and B subunit. It was clearly established that antisera to B subunit, devoid of significant anti-A subunit activity, neutralized the vascular permeability activity of cholera toxin. Antisera to A subunit contained neutralizing antibodies and antibodies to both A and B subunits. Absorption with B subunit removed both the toxin-neutralizing and anti-B subunit activities, while the anti-A activity was unaffected. Neutralizing antibody titers of rabbits immunized with B subunit were also observed to be significantly higher than neutralizing antibody titers of sera from A subunit-immunized rabbits, despite the overall similarity in anti-B subunit titers as determined by passive hemagglutination and radioimmunoassay of sera from the two groups of rabbits. Anti-α chain sera neither neutralized cholera toxin nor possessed significant antitoxin or anti-B subunit titers as determined by passive hemagglutination and radioimmunoassay. The anti-α chain sera contained high levels of antibody specific for A subunit, which is consistent with the hypothesis that the α chain is part of the A subunit structure. In contrast, the γ chain was not shown to be antigenic. Sera from convalescent cholera patients possessed toxin-neutralizing antibody as well as passive hemagglutination and radioimmunoassay antibody against both A and B subunits.

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