PRODUCTION AND TESTING OF ANTITOXINS 271 



without injuring the animal. Ligature of the vein after bleeding 

 is unnecessary. 



The cylinders and flasks are allowed to stand for two or three 

 days at or below 10 C. At the end of this time, the serum may 

 be pipetted or siphoned away from the clot and stored in the 

 refrigerator. In order to diminish the chances of contamination, 

 five-tenths per cent of carbolic acid or four-tenths per cent of tri- 

 eresol may be added. 



Antitoxin is fairly stable and if kept in a cool, dark place, may 

 remain active, with but slight deterioration, for as long as a year. 

 Kept in a dry state, m vacuo, over anhydrous phosphoric acid, by 

 the method of Ehrlich, it retains its strength indefinitely. 



Standardization. Antitoxin units being measured in terms of 

 toxin, uniformly of measurement necessitates the possession by the 

 various laboratories of a uniform toxin. Antitoxin being more stable 

 than toxin, uniformity of toxin is obtained by means of a standard 

 antitoxin distributed from a central laboratory. This was first done 

 by Ehrlich in Germany, and is now done for the United States by 

 the Public Health and Marine Hospital Service laboratories. Bottles 

 of the distributed antitoxin are marked with the number of units 

 contained in each c.c. Dilutions of this are mixed with varying 

 quantities of the toxin to be tested, the mixtures are allowed to 

 stand for 15 minutes to permit union of the two elements, and 

 injections into guinea-pigs of 250 grams weight are made. Thus the 

 L + dose of the toxin is determined. (The L + dose [p. 260] is the 

 quantity of poison not only sufficient to neutralize one antitoxin 

 unit, 5 but to contain an excess beyond this sufficient to kill a guinea- 

 pig of 250 grams in 4 to 5 days. L + is chosen rather than L , the 

 simple neutralizing dose, because of the difference between toxins 

 in their contents of toxoid and toxon. 6 ) 



The L + dose of the toxin having thus been determined, this 



5 The older definition of a unit of diphtheria antitoxin is the quantity of anti- 

 toxin sufficient to protect a guinea-pig of 250 grams against 100 times the fatal 

 dose of diphtheria toxin. This, however, holds true only if we are dealing with 

 normal toxins and antitoxins as at first devised by Behring. In the conditions 

 under which the measurements are made at present, however, this definition n.ust 

 be revised as follows: A unit of antitoxin is that amount of antitoxin which 

 will save the life of a guinea-pig if injected together with an L dose of the 

 toxin. 



' Madsen, in Kraus u. Levaditi, "Handbuch/ 7 etc., 1907. 



