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Preparation of Diphtheria Toxoid. The Action of Formaldehyde: Precipitation by Calcium

Augustus Wadsworth, James J. Quigley and Gretchen R. Sickles
The Journal of Infectious Diseases
Vol. 61, No. 2 (Sep. - Oct., 1937), pp. 237-250
Published by: Oxford University Press
Stable URL: http://www.jstor.org/stable/30089233
Page Count: 14
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Preparation of Diphtheria Toxoid. The Action of Formaldehyde: Precipitation by Calcium
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Abstract

In the experiments with crude diphtheria toxin here recorded the consumption of formaldehyde did not differ at incubator and room temperature. Because of the large proportion of amino compounds the results may be obscured by reactions with substances in the broth other than toxin. Detoxification takes place more rapidly and is completed earlier with the greater concentrations of formaldehyde at incubator temperature than at room temperature, and in purified as compared with crude toxin. The consumption of formaldehyde in the crude toxin treated with the larger concentration of formalin reached equilibrium before detoxification was complete, while with the small amount of formalin the consumption of formaldehyde paralleled the detoxification more closely. The antigenicity persisted as the toxicity became negligible and then diminished; it was greatest during the reversible stage of the toxoid reaction and diminished as the reaction became progressively irreversible. The rapidity of the change in antigenicity is also dependent upon the concentration of the excess free formaldehyde which may be removed by precipitation with calcium or alum at any stage of the reaction. The calcium is more reliable than the alum procedure which may yield preparations of variable potency and therefore requires preliminary standardization. In the toxin highly purified by ultrafiltration, the Kjeldahl nitrogen is reduced 98%; the Van Slyke, to the minimum detectable quantity. The reactions with formaldehyde, in contrast to the crude preparations, are more sharply defined. The consumption of formaldehyde, even in the presence of a large excess, is a minute quantity within the experimental error of the method, 1 to 3%. Neither the rate of consumption nor the amount, if any, that is consumed can be determined by present methods. Yet the reaction, directly dependent as it is upon the concentration of the reagents and the temperature, appears to be chemical in nature and, as indicated by changes in toxicity, reversible in a manner corresponding to the reaction of formaldehyde with certain amino acids. In the experiments with crude toxin there is evident a reversibility of the reaction of detoxification as well as a reversibility of a chemical reaction with formaldehyde that may take place with the toxin as well as with the amino compounds that are present. At 37 C. the reversibility of these reactions was demonstrable; at 6 C. it could not be detected. With the purified toxin containing scarcely detectable quantities of Van Slyke nitrogen, the action of formaldehyde appears to be directly on the toxin compound, possibly with an amino group, since the reversibility of detoxification was similar to that observed with formaldehyde and certain amino acids. In these experiments the action of formaldehyde does not appear to be dependent upon an intermediate reaction with a free amino acid. Reactions between formaldehyde and amino acid may be reversible or irreversible. When the reaction is reversible, formaldehyde may be set free to act upon the toxins. Detoxification of the purified toxin is inhibited when histidine is present in sufficient quantity to combine with the formaldehyde. The reaction between formaldehyde and histidine is not reversible. The crude diphtheria toxins prepared with different strains which require mediums that vary in composition detoxify and retain their antigenicity under conditions which, for practical purposes, must be carefully standardized with each toxin. Toxin produced by the strain no. 3203 in maltoseacetate medium was detoxified after treatment with 0.9% of formalin and incubation for 3 days at 36 to 37 C., followed by precipitation with calcium chloride. The preparation was superior in antigenic value to the toxoid prepared with 0.3% of formalin and incubation for 30 days at 38 to 39 C.

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