108 



INFECTION AND RESISTANCE 



against 100 times the fatal dose (M L D, minima dosis lethalis) 

 of toxin. Since the methods of antitoxin standardization employed 

 at present in the United States were worked out by Rosenau 10 along 

 the lines of Ehrlich's method, and the standard is based on the one 

 introduced by Ehrlich, the antitoxin unit as employed in this country 

 is identical with the one spoken of in the following paragraphs. 



In measuring the neutralizing value of antitoxin for toxin, then, 

 since both substances are chemically unknown and no purely chem- 

 ical indicator of neutralization is available, it was necessary to select 



a susceptible animal by means of which excess 

 of toxin, in mixtures of the two, could be de- 

 tected. As the standard test animal guinea 

 pigs of 250 grams were chosen, and improve- 

 ments in the methods of measurement were 

 introduced, in that the toxin and antitoxin, 

 instead of being separately injected as hereto- 

 fore, were mixed, allowed to stand for 15 to 

 30 minutes, and then injected together sub- 

 cutaneously. 



By means of this technique Ehrlich set 

 out to examine a large number of toxins and 

 their antibodies and obtained results which, 

 aside from their practical value, have had an 

 important influence upon the development of 

 the knowledge of antigen-antibody reactions. 

 These investigations were considerably 



complicated by the fact that neither the diphtheria toxin nor the anti- 

 toxin is very stable and deterioration occurs unless special methods of 

 preservation are employed. Since the antitoxin, however, is much 

 less unstable than the toxin, the former is employed in order to pre- 

 serve the standard, and is preserved in sealed U tubes (see Fig- 

 ure) with anhydrous phosphoric acid. Kept in this way, in black, 

 light-proof boxes, and at low temperature, it may be preserved for 

 months without appreciable loss of value and may be renewed by ac- 

 curate comparative measurements from time to time. This is carried 

 out for the United States, at the present time, by the Government 

 Hygienic Laboratories at Washington. 



Preservation of the toxin is much more difficult, and it is in 

 connection with the investigation of the instability of the toxin that 

 Ehrlich gained his first insight into the nature of the reaction. He 

 measured, in a number of toxic filtrates, the minimal lethal dose 

 for guinea pigs of 250 grams, establishing a time limit for death 

 in order to obtain more accurate comparisons. He designated as the 



10 Rosenau. U. S. P. H. & Mar. Hosp. Service. Hygienic Laboratory BulL> 

 21, April, 1905. 



TUBE FOR THE PRESERVA- 

 TION or THE STAND- 

 ARD ANTITOXIN. 



Taken from Eosenau, U. 

 S. Hygienic Labora- 

 tory Bulletin, No. 21, 

 1905, p. 53. 



