EDWIN J. BANZHAF 749 



tion of the toxin-antitoxin preparation as an indicator for immunes and non-immunes 

 in children under eight years of age in place of the Schick test. Considerable time is 

 saved, as about 85 per cent of the children of this age group are non-immune. One 

 mil is injected subcutaneously. In the non-immunes an erythema which may be ac- 

 companied with a slight tenderness and swelling at the site of injection develops in 

 twenty-four to forty-eight hours and gradually disappears. These non-immunes are 

 given at weekly intervals two more injections to complete the course of treatment. 

 Those that do not react to the first injection are immune and receive no further injec- 

 tions. Each mil of o.i L+ toxin-antitoxin preparation, i.e., each human dose, con- 

 tains 0.02 of a mil of aged-toxin broth and 0.0000375 of a mil of partially purified 

 antitoxin. 



EFFECTS OF FREEZING TOXIN-ANTITOXIN 



A reversible reaction caused by the solid freezing of a toxin-antitoxin mixture was 

 reported by Kelly' during January, 1924. 



The toxin in this mixture was prepared and used in the state of Massachusetts to prepare 

 a one L-|- toxin-antitoxin mixture. Samples of the mixture bearing the identification No. 

 loiC, which had not been frozen, were tested at the Hygienic Laboratory in Washington 

 and by the writer, and were found to be perfectly balanced for active immunization, as had 

 been shown by the pievious tests of White and Robinson^ in Massachusetts. 



In the Bureau of Laboratories, New York City Department of Health, samples of this 

 mixture No. loiC were frozen (Banzhaf and Greenwald)^ for eighteen hours at —8° C. and 

 on thawing were found to be toxic. Samples frozen for varying periods of time from twenty- 

 four hours to eleven days at a constant temperature of — 26° C. upon thawing were also found 

 to be toxic. The samples frozen for eleven days at —26° C. were considerably more toxic 

 than those frozen for eighteen hours at — 8° C. On retesting three weeks after freezing and 

 thawing they were found to be non-toxic. Upon refreezing a sample it became toxic again 

 and then again lost its toxicity. The hydrogen-ion concentration in this mixture before and 

 after freezing was pH 7.9. The sterility tests showed no growth. 



A reversible reaction was only obtainable by the writer when the lot of toxin No. 37Z 

 prepared in Massachusetts was used to prepare mixtures. A noticeable peculiarity of the 

 toxin and the toxin-antitoxin mixture sent to us from Massachusetts was that they had a 

 penetrating and persistent odor which presumably was due to an end-product developing 

 during the preliminary fermentation of the broth. Whether this indicates a possible connec- 

 tion with dissociation cannot be stated. Anderson and Leonard'' state that when antitoxic 

 serum or concentrated purified antitoxic serum (Gibson method) was used in balancing the 

 toxin for the toxin-antitoxin mixture, the mixture became toxic after freezing; but that when 

 the antitoxin prepared by the Banzhaf heating method for purifying was used, the mixture 

 did not become toxic after freezing. 



This does not clear up or explain the reversible reaction with the Massachusetts toxin. The 

 antitoxin used in balancing this toxin was prepared by the Banzhaf heating method. Kirkbride 

 and Dow5 found no increase in toxicity in their toxin-antitoxin mixture after freezing. The 



■ Kelly, F. L.: J.A.M.A., 82, 567. 1924. 



'White, B., and Robinson, E.: J.A.M.A., 82, 1675. 1924. 



3 Banzhaf, E. J., and Greenwald, C. K.: "The Effect of Freezing as Regards Toxicity on Diph- 

 theria Toxin-Antitoxin Mixture," read at the Buffalo meeting of the American Society of Immunoio- 

 gists, April, 1924. 



'« Anderson, J. F., and Leonard, G. F.: J. A.M. A., 82, 1679. 1924. 



5 Kirkbride, M. B., and Dow, J. E.: J. A.M. A., 82, 1678. 1924. ^ R^ 



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