Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

Abstract

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 antitetanus 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.

An improved whole cell pertussis vaccine with reduced content of endotoxin

“…Additionally, were also measured the potencies of the D and T components of DTP and DTPlow, by means of Toxin Binding assays. 20 , 21 Both preparations induced high and comparable levels of neutralizing antibodies against tetanus toxin (DTP 23.8 IU/ml and DTPlow 46.4 IU/ml; p = 0.27) and diphtheria toxin (DTP 66 IU/ml and DTPlow 32 IU/ml; p = 0.12), demonstrating that the LOS extraction had no effect on the immunogenicity against T and D antigens induced by the combined vaccine (data not shown).…”

Impaired Humoral Response to Vaccines among HIV-Exposed Uninfected Infants

“…In vitro tests for measuring tetanus and diphtheria antitoxin levels in serum were carried out by a standardized modified toxin-binding inhibition (TOBI) test as described by Hendriksen et al (13) but employing diphtheria toxoid instead of toxin (19,31,32).…”

Tetanus Immunity among Women Aged 15 to 39 Years in Cambodia: a National Population-Based Serosurvey, 2012

“…Serosurveys could be used to monitor progress toward the achievement and maintenance of MNTE given challenges of reliably assessing TT coverage through vaccination history (14). For in vitro assays of tetanus seroprotection, gold standards are the DAE (double-antigen enzyme-linked immunosorbent assay [ELISA]) and toxin binding inhibition (ToBI) tests, which are not commercially available (15)(16)(17)(18)(19). Indirect tetanus ELISAs exist, but they overestimate antibody levels in the low seroprotective range (0.01 to 0.2 IU/ml) (15,(19)(20)(21).…”