Antigenic relationships on the diphtheria toxin molecule: antitoxin versus antitoxoid

We used the mouse to produce antisera to native diphtheria toxin and diphtheria toxoid. With these antisera it was possible to distinguish between toxin and toxoid. By gel diffusion analysis, antitoxin detected antigenic determinants on toxin which were not available on toxoid, indicating that some determinants had been lost or altered by formalin treatment. Antitoxoid, on the other hand, showed reactions of identity between toxin and toxoid in gel diffusion. The toxin neutralization titers measured in tissue culture were the same for both antisera. Only antitoxin neutralized the adenosine 5'-diphosphate ribosyl-transferase activity of fragment A, but suprisingly both antisera had significant anti-fragment A titers when tested by passive hemagglutination. It is suggested that some of the anti-fragment A activity in antitoxin affects the enzyme active site, whereas that in antitoxoid does not, implying the existence of a least two independent antigenic regions on fragment A.