DIPHTHERIA TOXIN 105 



Almost invariably a bouillon filtrate contains toxones and toxoids besides the toxin. 

 For all practical purposes it is usual to consider an antitoxin unit as that amount 

 which will neutralize 100 M. L. D. (theoretically 200 units). Thus it would be 

 the amount which would neutralize 0.2 c.c. of the above noted toxin. In testing 

 filtrates as to their toxicity we make use of two limits, one designated Lo and the 

 other L+. When we add increasing amounts of a filtrate containing toxins and 

 toxones (the toxoids are less important because they do not show either the local 

 reaction or palsy of toxones and do not possess the acute death-producing power of 

 the toxin; they do, however, have combining power for antitoxin and are complicating 

 factors in standardization) to an antitoxin unit we gradually reach a point where the 

 slightest further increase will bring about slight reaction at site of inoculation of 

 the guinea-pig and possibly some slight paralysis. These symptoms are due to 

 toxone action. The amount of the toxic broth filtrate which is completely neu- 

 tralized by one antitoxin unit is called Lo. Upon further addition of the filtrate 

 to this Lo amount we finally reach a point when death of the 25o-gram guinea-pig 

 occurs in four days. This is called the L+ or fatal dose. Instead of having to add 

 only that amount of filtrate which is capable of killing the guinea-pig (i M. L. D.), 

 it is found that we must add an amount sufficient to kill 10 to 20 pigs. The explana- 

 tion is that while toxin and toxone both have power to combine with antitoxin and 

 to be neutralized, toxin has greater affinity and can dispossess toxone of its attach- 

 ment. Consequently when all the combining strength of the antitoxin unit (equal 

 to 100 M. L. D.) has neutralized the toxins and toxones added to it there is a complete 

 blocking of injurious action of toxins or toxones. When adding more filtrate to the 

 fixed amount of antitoxin a toxin molecule dispossesses a toxone molecule of its hold 

 on the antitoxin unit. The toxin is neutralized but a toxone is put in circulation 

 and is capable of causing reaction and palsy. Not until every toxone molecule has 

 been displaced can the further addition of i M. L. D., containing sufficient toxin to 

 kill, be free from the neutralizing antitoxin and capable of producing death in a 

 250-gram guinea-pig in four days. There have been prepared at the Hygienic 

 Laboratory in the U. S. and in various European laboratories, by laborious testing 

 of antitoxic serum, standardized antitoxin. By keeping dried antitoxic serum in a 

 vacuum tube under conditions preventing exposure to heat, light and moisture the 

 strength of the antitoxin unit remains stable. It may be stated that standardized 

 toxin soon tends to change in potency, therefore it is customary to take exactly 

 i unit of antitoxin and by adding to it increasing amounts of toxin to determine the 

 amount which will be sufiicient to kill the guinea-pig in four days. This is usually 

 somewhat over the amount which will kill 100 guinea-pigs, or 100 M. L. D., and is 

 designated the L+ dose of toxin. In practical application at biological product 

 institutions the L+ dose is tested with increasing amounts of antitoxic serum, as 

 drawn from the immunized horse, and that amount of serum which when mixed 

 with the L+ dose of toxin just allows death of the guinea-pig in four days is accepted 

 as one antitoxin unit. 



In the preparation of antitoxin horses are employed; the method being to inject 

 the bouillon filtrate or toxin subcutaneously at weekly intervals for a period of three 

 or four months. When each c.c. of the serum of the horse is found to contain about 

 250 to 500 antitoxin units the horse is bled from the jugular vein. Some sera contain 

 as much as 1300 units in a cubic centimeter. 



Methods of purifying and concentrating antitoxin are now employed by certain 



