Toxin-neutralizing Titers Research Articles

The titration of tetanus antitoxin III: A comparative evaluation of indirect haemagglutination and toxin neutralization titres of human sera

The tetanus antitoxin titres of 174 serum samples from healthy adults were determined by a standardization indirect haemagglutination test (IHA) and the conventional toxin neutralization (TN) test. The serum samples were titrated by the IHA test using glutaraldehyde-fixed and toxoid sensitized sheep erythrocytes before and after the treatment of the sera with 2-mercaptoethanol (2-ME). The IHA method has been found to be very sensitive and specific for the estimation of tetanus antitoxin in human sera. The IHA titres before the treatment of the sera with 2-ME were generally about four times higher than the TN titres and the correlation coefficient between these titres was 0.94. The IHA titres after the treatment of the sera with 2-ME were in good agreement with the TN titres and there was no statistically significant differences between the titres by the two methods. The tetanus antitoxin titres of 50% of the sera were below the minimum protective titres of tetanus antitoxin (0.01 IU/ml). In 19.5% of the sera the antitoxin level (IU/ml) ranged from 0.01 to 0.1, in 20.1% from 0.1 to 1.0 and in 10.4% from 1.0 to 10.0.

Influence of anti-fragment A of diphtheria toxin on passive hemagglutination with antitoxin.

Hemagglutination (HA) and toxin neutralization (TN) tests were used to titrate human and guinea-pig serum specimens taken at various stages of immunization for diphtheria antitoxin. The ratio of HA to TN titers varied significantly depending on the immune status. The ratios and the range of their variations became larger and high values exceeding five were often obtained after repeated booster immunization. Such a high value was proved to be related to the antibody against fragment A (FrA) of diphtheria toxin, since the ratio reduced significantly when anti-FrA was absorbed. Anti-FrA was not produced in children after the basic immunization with diphtheria toxoid and was detected in only one-third of vaccines after the booster injection given after 1 year. It was produced abundantly, however, when booster immunization was repeated. The pattern of production of anti-FrA in guinea pigs was similar to that in humans, when immunized with diphtheria toxoid with adjuvant. No anti-FrA was produced even when the animals were immunized repeatedly with plain diphtheria toxoid.

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.