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The Journal of Immunology, 1935, 29: 439-451.
Copyright © 1935 by The American Association of Immunologists, Inc.

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Purification of Diphtheria Toxin and Anatoxin

K. Ando and T. Komiyama

Hygienic Institute of the S. M. R. Co., Dairen, Manchuria

Abstract

1. Calcium-phosphate formed at a certain range of pH absorbs diphtheria toxin or anatoxin.
2. The largest percentage yield was obtained between pH 5.3 and 6.0 for the toxin used and between pH 5.6 and 6.3 for the anatoxin studied, whereas the purest product was obtained between 6.7 and 6.9 for the toxin and in pH 5.3 for the anatoxin so far as the Lf-unit was concerned.
3. For this purpose the use of H3PO4 and NaOH were found convenient.
4. By combining this method and the ZnCl2 method (Hosoya) it was possible to obtain anatoxin of high purity.
5. Anatoxins purified by these methods, after the addition of 0.5 per cent phenol, were found quite stable in storage at room temperature or in the cold.
6. Animal experiments with these purified anatoxins showed the same degree of antigenicity as that of the original anatoxin irrespective of their degree of purification, but in accordance with their Lf-units.
7. The anatoxin purified by the ZnCl2 method often showed no flocculation, as pointed out by Hosoya, yet its Lf-units could be determined by the blending method so that it is no exception to Ramon's conception that the antigenicity of anatoxin could be determined by the Lf-unit in vitro-test.
8. Contrary to the finding by Schmidt and Kjaer that the anatoxin purified by acid was less antigenic than that expected from its Lf-units, the antigenicity of the acid-purified anatoxin was found by the animal test to correlate with its Lf-units in vitro-test.
9. Highly purified anatoxins were compared with their original anatoxins by the Moloney test and it was found that the big difference in the N-content finds no expression in that test.







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