Immunochemical studies of diphtherial toxin and related nontoxic mutant proteins.
Competitive binding radioimmunoassays were used to analyze the immunochemistry of diphtherial toxin. Rabbit antisera obtained by immunization with formolized toxoid or fragment A were used to characterize purified toxin, toxoid, fragment A, and related nontoxic mutant proteins. Antitoxoid serum had a high titer of neutralizing activity. Most of the antibodies in antitoxoid bound to toxin but not to fragment A. The anti-fragment A antibodies that were present in antitoxoid recognized determinants of fragment A that were exposed on unnicked toxin. Formaldehyde treatment partially destroyed antibody-binding sites associated with the A and B domains of toxin. Anti-fragment A serum had a low titer of neutralizing activity. The specificities of the anti-fragment A antibodies in antitoxoid and anti-fragment A sera were different. Approximately half of the anti-fragment A antibodies in anti-fragment A serum recognized determinants of fragment A that were masked in toxin. Per unit of fragment A-binding activity, anti-fragment A serum was significantly more potent than antitoxoid serum as an inhibitor of the enzymatic activity of fragment A. By analyzing the antigenic structure of several nontoxic mutant proteins (cross-reacting materials) that cross-react with toxin, we distinguished three different subgroups of antigenic determinants associated with the B domain of toxin. Furthermore, the exposed antigenic determinants of the A domain of toxin were separated into two subgroups, both of which were distinct from the masked determinants of the A domain. The radioimmunoassays described here provide rapid, sensitive, quantitative, and versatile methods for immunochemical characterization of toxin or related cross-reacting proteins encoded by corynebacteriophages.
D000907:Antibodies, Bacterial / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis / Q000276:immunology; D001435:Bacteriophages / Q000276:immunology; D001667:Binding, Competitive; D003352:Corynebacterium / Q000276:immunology; D003429:Cross Reactions; D004166:Diphtheria Antitoxin / Q000032:analysis / Q000276:immunology; D004167:Diphtheria Toxin / Q000032:analysis / Q000276:immunology*; D004168:Diphtheria Toxoid / Q000032:analysis / Q000276:immunology; D000939:Epitopes / Q000032:analysis; D005557:Formaldehyde; D009500:Neutralization Tests; D011863:Radioimmunoassay
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D004166:Diphtheria Antitoxin; D004167:Diphtheria Toxin; D004168:Diphtheria Toxoid; D000939:Epitopes; D005557:Formaldehyde