Diphtheria toxin detoxification

Increasing the molecular weight of the aldehyde is reported as depressing the rate of detoxification and the firmness of union with diphtheria toxin (254). 

Detoxification. The process by which bacterial toxins are converted to harmless toxoids. Formalin is used to detoxify the toxins of both Corynebacterium diphtheriae and Clostridium tetani. The detoxification may be performed either on the whole culture in the fermenter or on the purified toxin after fractionation. 

The quality control of both diphtheria and tetanus vaccines requires that the products are tested for the presence of free toxin, that is for specific toxicity due to inadequate detoxification with formalin, at the final-product stage. By this stage, however, the toxoid concentrates used in the preparation of the vaccines have been much diluted and, as the volume ofvaccine that can be inoculated into the test animals (guinea-pigs)... 

The treated toxins are sometimes referred to as formol toxoids. Toxoid vaccines are very effective in the prevention of those diseases such as diphtheria, tetanus, botulism and clostridial infections of farm animals, in which the infecting bacteria produce disease through the toxic effects of secreted proteins which enzymically modify essential cellular components. Many of the clostridial toxins are lytic enzymes. Detoxification is also required for the pertussis toxin component of acellular pertussis vaccines. 

Detoxification. The process by which bacterial toxins are converted to harmless toxoids. Formaldehyde is used to detoxify the toxins of Corynebacterium diphtheriae, Clostridium botu-linum and Cl. tetani. The detoxification may be performed either on the whole culture in the fermenter or on the purified toxin after fractionation. Traditionally the former approach has been adopted, as it is much safer for the operator. However, the latter gives a purer product. The pertussis toxin used in acellular vaccines may be detoxified with formaldehyde, glutaraldehyde, or both, hydrogen peroxide or tetranitromethane. In the case of genetically detoxified pertussis toxin, a treatment with a low concentration of formaldehyde is still performed to stabilize the protein. 

In addition to Levy s studies referred to above (238, 239), the kinetics of detoxification of staphylococcus toxin by formaldehyde have been studied in some detail (252). The results show that the rate of reaction is directly proportional to the concentration of formaldehyde and to the square root of the hydroxyl ion. The reaction has an unusually high temperature coefScient of 4-5 for a change of 10 C. It is stated that the reaction follows a unimolecular course. This is true only when either the aldehydes or proteins are present in great excess, for the reaction should be bimolecular. Similar results have been obtained with diphtheria... 

C diphtheria vaccine is typical of a crude protein toxoid vaccine. Here the 58 kDa toxin is the antigen, and it is converted to a toxoid with formaldehyde and crudely purified. The cells are first separated from the toxin by centrifugation. Sometimes the pathogen culture is inactivated with formaldehyde before centrifugation. The supernate is treated with formaldehyde to 0.75%, and it is stored for 4 to 6 weeks at 37°C to allow complete detoxification (Pappenheimer, 1984]. The toxoid is then concentrated by ultrafiltration and fractionated from contaminants by ammonium sulfate precipitation. During detoxification of crude material, reactions with formaldehyde lead to a variety of products. The toxin is internally cross-linked and also cross-Hnked to other toxins, beef peptones from the medium, and other medium proteins. Because detoxification creates a population of molecules containing antigen, the purity of this product is only about 60 to 70%. 

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