THERAPEUTIC IMMUNIZATION IN MAN 461 



minimal lethal dose. This method has many points in its favor, and 

 has been recently utilized and improved upon by Homer. 



Eomer 30 31 32 has developed a method of diphtheria antitoxin 

 standardization which depends upon intracutaneous injections into 

 guinea pigs. The principle of this test consists in the observation 

 that, when very slight amounts of diphtheria toxin are injected intra- 

 cutaneously into the abdominal skin of guinea pigs, small areas of 

 local necrosis result within about 48 hours. When such injections 

 are made with mixtures of toxin and antitoxin the presence of free 

 toxin is indicated by the appearance of such necrosis. 



Before proceeding to the standardization by this method it ia 

 necessary to determine the "limes-necrosis" (just as Ehrlich deter- 

 mines his L+ dose), that is, the amount of toxin which, together 

 with a given amount of toxin (1/50, 1/200, or 1/2,000), will still 

 produce a minimal amount of necrosis after intracutaneous injection 

 into guinea pigs. It is necessary, therefore, arbitrarily to choose a 

 certain definite fraction of an antitoxin unit and mix this with vary- 

 ing amounts of toxin and inject the mixtures into guinea pigs intra- 

 cutaneously. Those mixtures in which the toxin is fully neutralized 

 will give rise to absolutely no lesion further than, possibly, a slight 

 local edema. Those in which there is a large excess of toxin will 

 cause extensive necrosis. Between the two, in the series, there will 

 be a mixture in which slight local necrosis results from the injection. 

 In this mixture the amount of toxin, just sufficient to cause notice- 

 able necrosis in spite of admixture with the antitoxin, contains the 

 L-n (limes necrosis) dose. 



When this has been determined, then unknown antitoxin can be 

 similarly measured against this L-n dose of the standard toxin. The 

 method has the advantage of permitting one to work with very small 

 quantities, since only a small fraction of a cubic centimeter need be 

 used for intracutaneous injections ; also it permits great economy of 

 animal material, since four or five tests can be simultaneously car- 

 ried out upon the abdominal wall of the same guinea pig. 



The technique is not easy. We have found in studying this 

 method in connection with some work carried on in our laboratory by 

 Dr. M. C. Terry, that a considerable amount of practice and experi- 

 ence is necessary, both in carrying out the procedure accurately and 

 in judging the lesions. However, when carefully and consistently 

 done by an experienced worker, this method gives results which cor- 

 respond with fair accuracy to measurements made of the same anti- 

 toxin by the Ehrlich method. This has been the experience of 

 Lewin, 33 and also of Terry in the few experiments carried out by him, 



30 Romer. Zeitschr. f. 1mm., Vol. 3, p. 208, 1909. 



31 Romer and Sames. Ibid., p. 344. 



32 Romer and Somogyi. Ibid., p. 433. 



33 Lewin. Centralbl. f. Bakt., Orig. Vol. 67, 1913. 



