TITRATION OF IMMUNE HEMOLYSIN 147 



dilutions decreases therefore not only the amount of hemolysin, the 

 quantitative estimation of which is the object of the experiment, but also 

 the complement. Inasmuch as the latter was not at first increased, a 

 point is soon reached where there is no complement at all in the diluted 

 fluid; as a result hemolysis cannot occur, for only the combination of 

 hemolysin + sufficient complement can exhibit any hemolytic action. 

 Correct titration consists therefore in allowing varying quantities of hemolysin 

 with a constant amount of complement to act upon a constant quantity of 

 red blood cells. The simplest method of accomplishing this is first to destroy 

 the complement by inactivation of the hemolytic serum, then to make the 

 desired dilutions, and finally to add, to all, the same amount of normal serum 

 as complement. The normal serum of an animal of the same species as 

 that which provided the immune serum can under no circumstances 

 serve as complement. On the contrary, foreign sera are much more 

 suitable; and guinea-pig's serum is especially recommended as comple- 

 ment when immune rabbit's serum is used. Not every complement serves 

 equally well for any immune serum. 



A very good hemolytic system and one which is almost exclusively 

 used for the complement fixation reaction, is sheep's blood as antigen, 

 rabbit's immune hemolysin as amboceptor and normal guinea-pig's serum 

 as complement. The preparation of these ingredients should be carried 

 out as follows: 



i. Sheep's Blood. This should be defibrinated and washed. Washing 

 is necessary because fresh sheep's blood contains complement; if the 

 blood is a few days old, washing is even more important. 



Although serum which is not fresh does not contain sufficient active 

 Comple- complement to cause the danger of superfluous complement, it never- 

 mentoids. theless contains substances which interfere with hemolysis. Probably 



the existence of " complementoids " is the disturbing factor. It must 

 be assumed that complement is composed of two biologically different parts, as is the 

 case with toxins and ferments. One is the haptophore group, which has affinity for 

 the complement ophile group of the amboceptor and is the more stable of the two. The 

 other corresponds to the energy group of the toxins (toxophore element) and of the 

 ferments. Just as after the destruction of the toxophore group there remain only 

 innocuous toxoids whose single perceptible activity consists in their ability to neutral- 

 ize antitoxins, so also, after the destruction of the weakly resistant energy elements of 

 the complement, there remain complementoids which lack the ability to activate a 

 bacteriolytic o"r hemolytic amboceptor, although by virtue of their uninjured hapto- 

 phore groups they bind the complementophile groups of the amboceptors. In this way 

 they usurp the place of whatever active complement may still be present, rendering 

 the latter inactive, and as a result hemolysis is absent or incomplete. 



Following the technique of Ehrlich and Morgenroth, a 5 per cent, sus- 

 pension of washed red blood corpuscles is employed to test a hemolysin. 



