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.     

Development of monoclonal latex diagnostic kit for diphtheria toxin and toxoid detection

Latex diagnostic kit with high specificity and sensitivity of diphtheria toxin and toxoid detection has been developed on the basis of protective monoclonal antibodies to diphtheria toxin and polyacrolein microspheres. Diagnostic kit was stable during 1 year of shelf-life (time of the study).     

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.     

Diphtheria toxin receptor. Identification of specific diphtheria toxin-binding proteins on the surface of Vero and BS-C-1 cells

The biochemical characteristics of specific receptor molecules for diphtheria toxin on the surface of two toxin-sensitive cell lines (Vero and BS-C-1) were examined. Diphtheria toxin was found to bind to a number of different proteins in Nonidet P-40 solubilized extracts of 125I-labeled cells. In contrast, permitting diphtheria toxin to bind first to labeled intact cells, which were subsequently solubilized and subjected to immunoprecipitation with anti-diphtheria toxin, resulted in a far more restricted profile of diphtheria toxin-binding proteins that possessed Mrs in the range of 10,000-20,000. Direct chemical cross-linking of radioiodinated diphtheria toxin to cell surface proteins resulted in the appearance of several predominant bands possessing Mrs of approximately 80,000. The Mr approximately 80,000 complexes were shown to be composed of radiolabeled diphtheria toxin (Mr 60,000) and unlabeled Mr approximately 20,000 cellular proteins. These complexes were judged to be a result of specific binding in that their appearance could be preferentially inhibited by the addition of a 100-fold excess of unlabeled diphtheria toxin. The formation of the Mr approximately 80,000 complexes was sensitive to prior trypsin treatment of the cells and to known inhibitors of diphtheria toxin binding. Furthermore, prior incubation of the cells with diphtheria toxin at 37 degrees C ("down regulation") markedly and specifically reduced the subsequent formation of the Mr approximately 80,000 cross-linked complexes, and these down-regulated cells were less sensitive to diphtheria toxin in cytotoxicity assays. Further incubation of down-regulated cells at 37 degrees C restored their ability to form Mr approximately 80,000 complexes; this regeneration requires protein synthesis and restores the cells' sensitivity to diphtheria toxin-mediated cytotoxicity. These results strongly suggest that a Mr 10,000-20,000 cell surface protein is, or constitutes a portion of, the functional diphtheria toxin receptor.