STANDARDIZATION OF DIPHTHERIA SERUM 79 



was done by Salomonsen and Madsen who by experimentation found 

 that the maximum height of the antitoxic curve was reached on the tenth 

 day after each inoculation. For this reason it is wise to choose this day 

 for the removal of the serum. As regards other sera, e.g., tetanus, different 

 periods have been empirically found to be most serviceable. As the 

 antitoxic curve does not remain at a high point for a long time, the 

 injections should be repeated from time to time. For highly immunized 

 horses, monthly injections usually suffice. 



After the serum has been obtained, the important problem which 

 arises is how to keep it sterile. This is accomplished by aseptic pre- 

 cautions at the time of obtaining the serum and eventually by the addition 

 of preservatives such as 1/2 per cent, carbolic acid or 0.4 percent, tricresol. 



This procedure finished, the next step is to estimate the amount of the 

 antitoxin content in the serum. 



According to v. Behring and Boer, the value of the serum should be 

 ascertained in respect to its: 



1. Protective power 1 . . r . 



against infection. 



2. Curative power J 



3. Protective power 1 . , . . . ,. 



against intoxication. 



4. Curative power ) 



v. Behring found that these four properties run parallel with each other 

 so that for practical purposes, it suffices to establish only one of these 

 qualities. For diphtheria serum it has proved most serviceable 

 Standardiza- to estimate the strength of the immunity against intoxica- 

 tion of tion, since one is dealing with a purely antitoxic serum. 

 Diphtheria 



Serum. Behring's original mode of standardization consisted in gradually add- 

 ing doses of serum to the minimal lethal dose of toxin and injecting the 

 mixtures into guinea-pigs, thus determining the smallest amount of serum capable of 

 preventing death of the animal. It was soon found, however, that this method gave 

 too inconstant results because the individual minimal lethal dose was too variable. 



Ehrlich, therefore, modified the process by using ten times the minimum lethal dose. 

 This amount of toxin, mixed with decreasing amounts of serum and made up to 4 c.cm. 

 with physiological salt solution was injected subcutaneously into a guinea-pig. The 

 smallest amount of serum which saved it from being killed on the fourth to fifth day was 

 thus .estimated. 



The method of standardization used at the present time owes its origin 

 to Ehrlich. 



In order to attain uniformity in the comparative value of all sera, 

 Behring and Ehrlich recommended the adoption of two empirical values; 

 "the normal toxin/' and the " normal curative serum." 



The normal diphtheria toxin is one which contains enough toxin in i 

 c.cm. to kill 25,000 gms. of guinea-pigs or 100 guinea-pigs each weighing 

 250 gms. 



