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

M.H Sonobe,H.G Higashi,D Sakauchi,A.G Trezena,F Fratelli,J.F Morais,V.L Takano,F.B Guilhen,S.M.A Prado

doi:10.1590/s0100-879x2007000100009

M.H Sonobe, H.G Higashi + Show 7 more

Open Access

https://doi.org/10.1590/s0100-879x2007000100009

Copy DOI

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 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.

Highlights

In vivo and in vitro tests for measuring tetanus and diphtheria antitoxin levels in serum from different species have been standardized and executed for many years
The toxin-binding inhibition (TOBI) test reaction is based on the capacity of the coated antitoxin in the second step (P2) to bind any anatoxin that did not interact with the antibodies of the sera tested in the first step (P1)
We describe here a second modification of the TOBI test protocol [10,11], denominated Modi-TOBI test, in order to detect low levels of tetanus and diphtheria antibodies in serum

Summary

Introduction

In vivo and in vitro tests for measuring tetanus and diphtheria antitoxin levels in serum from different species have been standardized and executed for many years. There is the neutralization test in microcell culture using VERO (green monkey renal epithelium) cells [2,3]. Several in vitro serologic methods have been described, such as passive hemagglutination [1,4,5,6], enzymelinked immunosorbent assay (ELISA) [1,79] and the toxin-binding inhibition (TOBI) test [10,11]. This test involves high costs, is time-consuming, and requires specialized personnel (trained to work with toxins), as well as large numbers of animals and significant amounts of serum. In vitro serologic methods have been developed as alternative approaches to reduce costs and to improve animal welfare

Methods

Results

Conclusion