THB CONSTITUENTS OF DIPHTHERIA TOXIN. 485 



If, therefore, the affinity between diphtheria toxin and antitoxin 

 is so great, we shall have to ascribe the irregular course of the neutraliza- 

 tion process to other factors thai: those assumed by Arrhenius and 

 Madsen. 



Diphtheria Toxins. 



In order to understand what follows it will be necessary to speak 

 of some of the main principles of toxin-antitoxin analysis. As is 

 well known diphtheria toxin is the bouillon fluid in which the diph- 

 theria bacilli have grown, and to which they have given up their 

 toxic secretory products. In order to determine the toxicity we 

 make use of guinea-pigs. The lethal dose (L. D.) is that amount 

 of poison which will surely kill a guinea-pig weighing 250 grammes 

 on the fourth day. In order to determine the relations between toxin 

 and antitoxin it is best to start from the serum because this can be 

 preserved constant by means of the methods devised by me (vacuum, 

 drying). This dry serum also serves as the standard for the officia 

 titration. The immune unit (I. E. = Immunitats Einheit) is, of course, 

 an arbitrary quantity which originated by terming that amount of 

 antitoxin a unit which just neutralized 100 L. D. of a poison that 

 happened to be available at the time, so that the mixture when injected 

 did not produce even the slightest trace of illness (either general or 

 local reaction). 



If one mixes one immune unit of serum with graduated amounts 

 of poison, two limits may be obtained. One of these is termed 

 limit zero (L ), and corresponds to the quantity of poison which is 

 completely neutralized by 1 I. E. The other is limit death (Lf) and 

 corresponds to that quantity of poison which on the addition of 1 I. E' 

 is so far neutralized that only just one L. D. remains. Of these two 

 limits the Lf is very easily and accurately determined so that it 

 serves as a measure in testing the potency of the diphtheria serum. 

 This limit signifies nothing more than that of x L. D. present, 1 I. E. 

 neutralized x\ L. D., so that just 1 L. D. remains free and leads 

 to the death of the guinea-pig in four days. 



A priori one might have expected that the number of lethal doses 

 which are neutralized by 1 I. E. is always the same in poisons from 

 different sources. The only difference which one would have ex- 

 pected would be that in different poison solutions, the volume in 



