ELISA for titration of antibodies to tetanus toxoid in sera of immunized guinea pigs as an alternative to the toxin neutralization test in mice

Abstract

A paper by Manghi et a!. (1994) on the 'Devel­ opment of an ELISA for measuring the activity of tetanus toxoid in vaccines and comparison with the toxin neutralization test in mice' prompts this response. A major problem with the paper is that the authors have confused methods of testing the potency of tetanus toxoid (IT) as per the British Pharmacopoeia (1973), the British Pharma­ copoeia (1980) and the United States Minimum Requirements (1952). The three methods are very different. The British Pharmacopoeia (1973) method, which is no longer employed in Great Britain, is based on two sequential injections of IT into guinea pigs. This method has been fol­ lowed by Manghi et al. (1994) who refer to it as the British Pharmacopoeia (1980) method. The British Pharmacopoeia (1980) method of potency testing of adsorbed TT, which is currently used in most European countries including Great Britain and which is recommended by the World Health Organization (1990), does not involve titration of sera from immunized guinea pigs. This method requires a single injection of several dilutions of test and reference vaccines into groups of guinea pigs or mice and challenging the immunized ani­ mals after 28 days with tetanus toxin. The po­ tency of the test vaccine is determined against the reference vaccine by calculating 50% immunizing doses of both test and reference vaccines. The British Pharmacopoeia (1973) method for ad­ sorbed IT requires two injections of nine guinea pigs with 1/10 of a single human dose with a 4 week interval and bleeding of guinea pigs not later than 2 weeks after the second injection. The vaccine passes the test if at least 113 of guinea pigs show tetanus antitoxin levels in their sera not less than 0.5 IUIml or at least 2/3 of guinea pigs show tetanus antitoxin levels not less than 0.05 IU1m!. Manghi et a!. (1994) injected guinea pigs by the British Pharmacopoeia (1973) method and followed the requirements of the US potency method which uses a different immunization schedule. The US method requires injection of guinea pigs with a single dose of half of the total human dose (1.5 times a single human dose) and bleeding the guinea pigs not later than 6 weeks afterwards. The vaccine passes the US minimum requirements if the pooled sera of at least four guinea pigs show no less than 2 IUIml of tetanus antitoxin. It has been shown that the affinity of antibod­ ies elicited in guinea pigs after injection of diph­ theria toxoid (Gupta et aI., 1994) and tetanus toxoid (Gupta and Siber, 1994b) depends upon the immunization status of animals. In the experi­