The objective of this study is to develop a suitable in vitro potency test for the tetanus component in biological products and to correlate this with the official test performed in guinea pigs. An ELISA has been developed to measure the amount of tetanus neutralizing antibodies in serum from guinea pigs immunized with adsorbed biological products containing the tetanus toxoid component. A microtiter plate is coated overnight with chromatographically purified tetanus toxoid. The coated plates are then blocked with BSA for four hours. The serum (primary antibody) being tested is diluted two-fold starting at 1/500, added to the prepared plate. and incubated overnight. An anti-guinea pig alkaline phosphatase conjugate (secondary antibody) is added to the plate and is incubated for four hours. Finally, alkaline phosphatase substrate is added to the plate and incubated for 15 minutes. The plate is read at 410 nm and analyzed by a computer program (the parallel line assay) designed specifically for this application. The unitages obtained from this assay are compared with those values determined in the official potency test in guinea pigs. Standardization of this assay is being considered by incorporating both the international and U.S. Reference preparations. Optimization of this assay is nearing completion. In addition to the ELISA test, a TOBI test specific for tetanus is being explored. During the developmental process of the TOBI test, reagent development and purification is achieved using animals for antibody production, chromatography (affinity, ion-exchange, and gel filtration) for purity, EIA's for activity and Biorad assay for protein concentrations. The method of enzyme conjugation to a specific tetanus antibody will be performed using dialysis and enzyme launches. An additional study on the neutralization activities of IgG subclasses in Guinea Pigs is in the initial stages of development.
