PRACTICAL APPLICATIONS 375 



After determining the unit of amboceptor or complement that is, 

 after adjusting the hemolytic system to exact proportions the results 

 that follow the varying quantities of complement and amboceptor re- 

 quire the most careful consideration. Less than one unit of amboceptor 

 with one unit of complement cannot yield complete hemolysis; likewise 

 if with one unit of amboceptor less than a unit of complement is combined 

 hemolysis is incomplete; with one unit of amboceptor and one unit of 

 complement and a double dose of corpuscles hemolysis will also be in- 

 complete. With less than a unit of complement and an excess of ambo- 

 ceptor, however, hemolysis may be complete. The complement may be 

 reduced to so small an amount that hemolysis is incomplete no matter 

 how much amboceptor is used, but the important fact to be borne in 

 mind is that a slight decrease in complement may be compensated for 

 by the presence of many units of amboceptor, so that complete hemolysis 

 results and a false reaction is secured. The converse of this is true to a 

 less marked extent i. e. } an excess of complement may compensate for 

 a decrease in amboceptor, but is less capable of doing so. 



These facts are of the utmost importance in making hemolytic ex- 

 periments, as in complement-fixation reactions, where the entire test 

 depends upon demonstrating whether or not a portion or the whole of 

 the complement used has been fixed. Unless, in a series of hemolytic 

 reactions, the amount of amboceptor employed is the same throughout, 

 the amount of complement acting in these cannot be determined by 

 comparing the degree of hemolysis. This is true especially in cases 

 where a small amount of complement is fixed, as in the Wassermann re- 

 action, with a serum containing a small amount of syphilitic antibody, 

 when the presence of an excess of hemolytic amboceptor may give 

 complete hemolysis and overshadow the fact that a small amount 

 of complement has been actually fixed by syphilitic antibody and an- 

 tigen. 



Method of Titration of Immune Hemolysin. Various methods have 

 been employed by different workers in this field, but all are based upon 

 the same principles as have been here outlined. 



A small amount of immune serum is inactivated by heating in a water- 

 bath at 56 C. for half an hour. In testing the serum of a rabbit during 

 the process of immunization 2 or 3 c.c. of blood are easily secured from 

 the ear, and the serum is separated. After it has been inactivated, the 

 serum is diluted to 1 : 100 (1 c.c. of serum to 99 c.c. of salt solution, or 

 0.1 c.c. serum-f-9.9 c.c. of salt solution). 



Fresh guinea-pig serum is secured for complement by bleeding a 



