Development of Murine Monoclonal Antibodies, Characterization and Quantification of Diphtheria Toxoid in Vaccine Batches

Diphtheria is caused by Cornybacteria diphtheria and it is one of the contagious diseases with a mortality rate of 5% to 10% worldwide. Though the mass immunization of diphtheria vaccine reduces the mortality, but the immediate effective treatment for this disease involves the administration of anti-diphtheria polyclonal antibodies or diphtheria antitoxin (DAT) produced from Equines. However the availability of antidiphtheria polyclonal antibody is very limited due to the less number of manufacturers. Hence the development of monoclonal antibodies (mAbs) with neutralizing capability may act as a potent alternate candidate to DAT or as an effective therapeutic agent. In the present study, we have developed and characterized five mouse monoclonal antibodies against diphtheria toxoid. The specificity of the mAbs was established by its non-reactivity towards other toxins by indirect ELISA (Enzyme linked Immunosorbent assay) and competitive ELISA with the commercial mAb. The non-competing mAbs were used to develop immunocapture ELISA for the quantification of toxoid content in in-process samples during manufacture of the vaccine. The r² value obtained by the regression analysis was 0.996. This ELISA can be adapted to measure the toxoid content and blending of the vaccine can be performed based on the estimated toxoid. The neutralizing activity of the mAbs against diphtheria toxin was performed by in vitro cell based neutralization assay using Vero cells. The cytotoxicity assay demonstrated that mAb neutralized the toxin in a concentration dependent manner. We have further shown that the mAb binds to the receptor binding domain of diphtheria toxin and it blocks the toxin from binding to the heparin-binding epidermal growth factor like growth factor by ELISA. These monoclonal antibodies may have a potential in development of therapeutics and diagnostics.