Quantitative estimation of diphtheria and tetanus toxoids. 4. Toxoids as international reference materials defining Lf-units for diphtheria and tetanus toxoids

The Lf-unit, which is used in the control of diphtheria and tetanus toxoid production and in some countries also to follow immunization of horses for production of antitoxins, has hitherto been defined by means of antitoxin preparations. A diphtheria toxoid and a tetanus toxoid preparation, both freeze-dried, were examined in an international collaborative study for their suitability to serve as reference reagents in the flocculation tests and for defining the Lf-units. It was shown that flocculation tests using the reference toxoids are very reproducible and reliable and the WHO Expert Committee on Biological Standardization established: the toxoid called DIFT as the International Reference Reagent of Diphtheria Toxoid for Flocculation Test with a defined content of 900 Lf-units of diphtheria toxoid per ampoule; and the toxoid called TEFT as the International Reference Reagent of Tetanus Toxoid for Flocculation Test with a defined content of 1000 Lf-units of diphtheria toxoid per ampoule.     

Comparison of immune responses to diphtheria and tetanus toxoids of various mouse strains

Immune responses of 11 mouse strains with known genetical characteristics and two outbred strains to diphtheria and to tetanus toxoids were compared. Both diphtheria and tetanus antitoxins were titrated by passive hemagglutination. From the pattern of the immune response, the mouse strains tested may be classified into four groups. [1] Strains ddY (SPF) and ddY (conv) and those with haplotype H-2b, such as C57BL/6 and C57BL/10, were high responders to both toxoids. [2] Strains with H-2d, such as BALB/c, B10.D2 and DBA/2Cr, were intermediate responders to both toxoids. [3] Strains with H-2k, H-2a or, H-2m, such as C3H/He, B10.BR, B10.BR/SgSn, B10.A/SgSnJ and B10.AKM/O1a, were high responders to diphtheria toxoid but low responders to tetanus toxoid. [4] The strain with H-2h4, B10.A (4R), was a poor responder to both toxoids.     

Calibration of replacement international standards of diphtheria and tetanus toxoids for use in flocculation test

The 1st International Reference Reagents (IRR) of Diphtheria and Tetanus Toxoids for Flocculation Test (DIFT and TEFT) were established by the WHO in 1988. These reagents are essential for the standardization of assays used to calculate Lf units of toxoids. Candidate replacement materials were provided by several European vaccine manufacturers and were formulated and freeze-dried at NIBSC. This paper provides a summary of the results of an international collaborative study including 18 laboratories from 16 countries, which examined the candidate replacement materials in a variety of methods. Materials 02/176 and 04/150 were proposed and adopted by the Expert Committee on Biological Standardization of WHO in October 2007 as 2nd WHO International Standards of Diphtheria and Tetanus Toxoid for use in Flocculation Test. The replacement standards were assigned the value of 1100 and 690Lf/ampoule, respectively, based on results of flocculation tests carried out using provided reagents. Material coded 02/176 fully complied with the WHO specifications for stability, residual moisture content, precision of fill and sterility. Stability of material coded 04/150 was slightly lower than expected but predictions were based only on 2-year data and were to be further monitored, post-adoption.     

Single radial immunodiffusion as a method for the identification and quantitation of diphtheria and tetanus toxoids in adsorbed vaccines

The immunochemical techniques of double diffusion and single radial diffusion in agarose gels were compared and each considered as possible alternative methods to the methods stipulated by the European Pharmacopoeia for the identification of diphtheria and tetanus toxoids in adsorbed vaccines. Both methods identified the toxoids but single radial diffusion was found to be preferable as the precipitin bands formed were visible without staining. Single radial diffusion was further investigated for its suitability as a quantitative method and was found to give reproducible estimates of the amount of toxoid present in all vaccines tested. However, in the case of tetanus toxoids these estimates were lower than the amounts stated to have been incorporated in the vaccines by the manufacturers. It was concluded that single radial diffusion would be a suitable replacement in the European Pharmacopoeia as a method for the identification of the diphtheria and tetanus components of adsorbed vaccines provided that elution could also be achieved from vaccines containing calcium phosphate.     

Dynamic changes of horse serum T-globulin immunization with snake venoms, tetanus and diphtheria toxoids

In course of immunizing horses with snake venoms, tetanus and diphtheria toxoids, a new serum component, T-globulin, was formed and migrated between the beta- and gamma-globulins. The T-globulin content was parallel with the antibody titre after the middle course of immunization. There were many components in snake antivenin and T-globulin was composed of most of those components. The components of diphtheria T-globulin were the same as those of crude antitoxin and tetanus T-globulin except one precipitin.     

Comparative quantitation for the protein content of diphtheria and tetanus toxoids by DC protein assay and Kjeldahl method.

DPT, a combination vaccine against diphtheria, tetanus and pertussis is available since many years and still continued in the national immunisation schedule of many countries. Although highly potent, reactions to DPT vaccine are well known, mainly attributed to the factors like Pertussis component, aluminum adjuvant and lower purity of tetanus and diphtheria toxoids. The latter most important aspect has become a matter of concern, specially for the preparation of next generation combination vaccines with more number of antigens in combination with DPT.Purity of toxoid is expressed as Lf (Limes flocculation) per mg of protein nitrogen. The Kjeldahl method (KM) of protein nitrogen estimation suggested by WHO and British Pharmacopoeia is time consuming and less specific. Need has been felt to explore an alternative method which is quick and more specific for toxoid protein determination. DC (detergent compatible) protein assay, an improved Lowry's method, has been found to be much more advantageous than Kjeldahl method.     

Vitamin A as adjuvant to the mouse immune response to pertussis, tetanus and diphtheria vaccines

Vitamin A was used as adjuvant, comparatively with Al(OH)₃, in pertussis, tetanus and diphtheria vaccines. Both groups induced a primary immune response in mice, and one single booster dose elevated the antibodies titers in average 554 times to vitamin A groups and 104 times to Al(OH)₃. These antibodies titers correlate with sera IL-4 in immunized animals, suggesting a Th2 response. Other cytokines detected in the sera and/or lymphocytes culture supernatants (IL-2 and IFN-) indicated that vitamin A could also modulate a Th1 response in DPT and acellular pertussis vaccines.     

The quantitative estimation of diphtheria and tetanus toxoids. 1. The flocculation test and the Lf-unit

Considerable confusion exist in the quantitative estimation of diphtheria and tetanus toxoids. The present paper is the first in a series of papers comparing various quantitative methods. It describes the flocculation test, gives a mathematical model for the three-dimensional reaction surface, and suggests different designs which facilitate the unbiased calculation of the end-point.     

Liposomes as immunological adjuvants for tetanus vaccine

Finding a new immunological adjuvant has been a challenge all along. The aim of our study is to improve the level of antibodies against tetanus using liposomes as adjuvant. As can be seen from the results of our study, the liposomes are a competitive candidate in this respect.     

Quantitative estimation of diphtheria and tetanus toxoids. 3. Comparative assays in mice and in guinea-pigs of two tetanus toxoid preparations

Two freeze-dried international reference tetanus toxoids of different origin and purified by different methods were compared in various potency assay systems, in vitro as well as in vivo. When the antigenic contents in the two toxoids are used as the basis for expressing the relative potency, different tests in animals gave different potencies. It is concluded that, as a result of such differences, tetanus vaccines can hardly be quantitated unambiguously in potency assays in animals. Since, however, a biological immunogenicity control seems necessary, a more simple type of test is suggested, which will require much less resources in terms of animals and manpower.     

Quantitative estimation of diphtheria and tetanus toxoids. 5. Comparative assays in mice and in guinea-pigs of two diphtheria toxoid preparations

Two freeze-dried international reference diphtheria toxoids of different origin were compared in biological assays in guinea-pigs and mice under different adjuvant conditions. When the antigenic content in the two toxoids was used as denominator for determination of relative potency, that is to say quantitation of immunogenic power per unit amount of antigen, the design of the animal assay proved to have a major influence. Similar observations have been made previously also for tetanus vaccines. It is concluded that diphtheria vaccines as well as tetanus vaccines can hardly be quantitated unambiguously using the currently recommended potency assays in animals. A new scheme for control of toxoid vaccine production is suggested, with more emphasis on the control of the bulk purified toxoid, which would make the release of final products more simple and rapid.