title: Immunochemical studies of diphtherial toxin and related nontoxic mutant proteins.
abstract: 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.
mesh_terms: 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
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D004166:Diphtheria Antitoxin; D004167:Diphtheria Toxin; D004168:Diphtheria Toxoid; D000939:Epitopes; D005557:Formaldehyde