Development of ELISA (TOBi) assay for determination of Tetanust toxoid potency

One of the major uses of large number of laboratory animals in manufacturing the vaccines is the quality control testing of vaccines, particularly potency testing of vaccines containing the tetanus toxoids by either lethal challenge or serum neutralization tests. Recently, because of various difficulties to obtain quality laboratory animals and in adequate environmental conditions, a lot of efforts have been made (such as those of World Health Organization’s) to attempt to limit the use of animals (in vivo) in the research in routine quality control of human and veterinary vaccines by in vitro assays such as Toxin Binding Inhibition (ToBi) assay. ToBi assay is based on the inhibition of the binding of toxin to an antitoxin-coated ELISA plate which detects the free toxin into the toxin-serum mixtures. In this study we tried to demonstrate and setting up the parameters of ToBi assay for the tetanus toxoid potency determination compared with serum neutralization test.
Groups of mice were immunized with several of dilutions of reference or test tetanus toxoid vaccines and groups of guinea pigs were also immunized (for Serum Neutralization test) with the dose of half of total human dose of tetanus toxoid vaccines manufactured by Razi Vaccine & Serum Research Institute. Serum samples were pooled and titrated for levels of tetanus antitoxin by toxin binding inhibition (ToBi) test and by the serum neutralization (SN) test in mice. The ToBi test was carried out by making of Serum dilutions and a fixed amount of tetanus toxin added to each serum dilution. After incubation, the mixtures were transferred on to ELISA plate coated with purified equine anti-tetanus IgG. Then free toxin is detected by addition of HRP labelled equine anti-tetanus IgG.
The results were shown to be significantly correlated. The consistency between the ToBi test derived from the in vitro data and from the in vivo data were reliable. The results ranged from 1.8 to 5.3 IU/ml for tetanus toxoid vaccines by ToBi test and 3.3 to 6 IU/ml for tetanus toxoid vaccines by SN in mice. The ToBi test may able to distinguish between highly potent and less potent vaccines.
It is concluded that the ToBi test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved. The ToBi test offers distinct advantages in relationship to serum neutralization in mice and lethal challenge tests.