PRODUCTION AND TESTING OF ANTITOXINS 221 



It is necessary to determine the strength of the poison. This is done 

 according to Behring l by determining the smallest amount of toxin 

 which will kill a white mouse of twenty grams weight within four days. 

 This is most easily done by making dilutions of the toxin ranging from 

 1 : 100 to 1 : 1,000, and then injecting quantities of 0.1 c.c. of each of 

 these dilutions subcutaneously into white mice. In this way, the mini- 

 mal lethal dose is ascertained. ' 



For the actual production of antitoxin, horses have been generally 

 found to be the most favorable animals. The horses should be healthy 

 and from five to seven years old. The first injection of toxin admin- 

 istered to these animals should be attenuated in some way. Vari- 

 ous methods for accomplishing this have been in use. In America, 

 the first injection of about ten to twenty thousand minimal lethal doses 2 

 (for mice of twenty grams weight) is usually made subcutaneously to- 

 gether with sufficient antitoxin to neutralize this quantity. In Germany, 

 v. Behring uses, for his first injection, a much larger dose of toxin to 

 which about 0.25 per cent of terchlorid of iodin has been added. 

 Immediately after an injection, the animals will usually show a reac- 

 tion expressed by a rise of temperature, refusal of food, and some- 

 times muscular twitching. A second injection should never be given 

 until all such symptoms have completely subsided. This being the case, 

 after five to eight days double the original dose is given together with 

 a neutralizing amount of antitoxin or with the addition of terchlorid of 

 iodin. Again after five to eight days, a larger dose is given and there- 

 after, at similar intervals, the quantity of toxin is rapidly increased. In 

 America the neutralizing antitoxin is omitted after the third or fourth 

 injection; in v. Behring's laboratory the quantity of terchlorid of iodin 

 is gradually diminished. The increase of dosage is often controlled by 

 the determination of the antitoxin contents of the animal's blood serum. 

 The immunization is increased until enormous doses (500 c.c.j of a 

 toxin in which the minimal lethal dose for mice is represented by 

 0.0001 c.c., or less, is borne by the horse without apparent harm. 



The antitoxic serum is then obtained by bleeding from the jugular 

 vein, as in the case of diphtheria antitoxin. It may be preserved in the 

 liquid state by the addition of five-tenths per cent of carbolic acid or 

 four-tenths per cent of tricresol. 



1 v. Behring, Zeit. f. Hyg., xii, 1892 ; Deut. med. Woch., 1900. 



2 According to Park the "horses receive 5 c.c. as the initial dose of a toxin 

 of which 1 c.c. kills 250,000 grams of guinea-pig, and along with this a sufficient 

 amount of antitoxin to neutralize it." 



