Suitability of the Vero Cell Method for Titration of Diphtheria Antitoxin in the United States Potency Test for Diphtheria Toxoid

Abstract

Abstract. The in vitro Vero cell method for titration of diphtheria antitoxin in immunized guinea pig sera was standardized to obtain comparable results to the in vivo toxin neutralization (TN) test in guinea pigs used to test potency of adsorbed diphtheria toxoid according to the United States Minimum Requirements. In the Vero cell method, the antitoxin titers of the guinea pig sera, obtained 4 weeks after immunization, were markedly dependent on the toxin dose level used in the assay. A toxin dose level termed the Lcd/1 dose ( limit of cytopathic dose at 1 IU/ml) for Vero cell method gave comparable estimates of antitoxin activity to the in vivo TN method performed at L+/1 dose of toxin. The Lcd/1 toxin dose level for the Vero cell method is defined as the minimum concentration of diphtheria toxin required to produce a cytopathic effect on Vero cells in 4 days when the solution of diphtheria toxin is mixed with an equal volume of standard diphtheria antitoxin whose concentration is 1 IU/ml. When lower dose levels of toxin were used in the Vero cell assay (Lcd/10 to Lcd/1000), the concentrations of antitoxin in 4 weeks guinea pig sera were 2 to 11·7 times lower than with the Lcd/1 dose level and similarly lower than those measured by the in vivo TN test at L+/1 level. In contrast, the concentration of antitoxin measured in sera from guinea pigs who have been boosted with diphtheria toxoid increased approximately two-fold with the Lcd/1000 dose level. The most likely reason for the differences between sera of primary and secondary immunizations is that the former sera are of much lower avidity and the latter of slightly higher avidity than the equine antitoxin standard. Low affinity antibodies bind antigen less well at low reactant concentrations. We propose that the Vero cell method at the Lcd/1 level deserves further evaluation to determine whether it can replace the in vivo TN method used for titration of diphtheria antitoxin in the U.S. potency test for diphtheria toxoid, thereby saving animals, cost and labor.