A method for the screening of
tetanus and
diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of
tetanus or
diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with
tetanus toxoid,
diphtheria-
tetanus or triple
vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard
tetanus or
diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with
tetanus or
diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a
peroxidase-labeled
tetanus or
diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard
antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-
tetanus detection, or in guinea pigs for anti-
diphtheria detection. Both assays were suitable for determining wide ranges of
antitoxin levels. The linear regression plots showed high correlation coefficients for
tetanus (r(2) = 0.95, P < 0.0001) and for
diphtheria (r(2) = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of
neutralizing antibodies, thus permitting the in vitro control of serum
antitoxin levels.