422 PHENOMENON OF COMPLEMENT FIXATION 



its amboceptor, the addition of too large an amount of hemolysin may 

 result in complete hemolysis of the corpuscle, and thus overshadow the 

 slight but specific fixation of complement. 



On the other hand, hemolysis cannot be complete if the dose of 

 amboceptor is too small. With a given dose of corpuscles and comple- 

 ment a certain amount of hemolysin is necessary to produce hemolysis, 

 this dose being determined by a process, of titration, as described in a 

 previous chapter. If less than this dose is used, but the amounts of 

 corpuscles and complement remain the same, hemolysis will be corres- 

 pondingly incomplete and lead to false positive reactions. 



A very important feature of all complement-fixation tests will be 

 seen to be a proper and accurate adjustment of the hemolytic system. 

 Taking arbitrary amounts of corpuscles and hemolysin as constants, 

 the quantity of complement necessary to produce hemolysis may be 

 determined (titration of complement); or, taking corpuscles and com- 

 plement as constants, the amount of hemolysin necessary to effect 

 complete hemolysis may be determined. One or the other or both titra- 

 tions should be made before the main test is attempted, in order to 

 avoid using an excess or too little of either ingredient. If the exact unit 

 of complement and hemolysin are used, the results must be very care- 

 fully guarded, because in a general way all antigens and serums exert a 

 slight anticomplementary action that may yield results that will be 

 interpreted as weak positive reactions. For this reason the original 

 complement-fixation tests invariably called for a slight excess of com- 

 plement or hemolysin or both, to allow for possible non-specific com- 

 plement fixation, and this is a good general rule that makes the reaction 

 somewhat less delicate, but more reliable in the long run, especially for 

 inexperienced workers. 



Complements of different species of animals act differently in activat- 

 ing a hemolytic amboceptor and to ward fixation by antigen-antibody com- 

 binations. For instance, a complement from one" animal may readily 

 enough combine with a hemolytic amboceptor to produce hemolysis, 

 but will not lend itself for fixation, and is, therefore, unfit for comple- 

 ment-fixation tests. Noguchi and Bronfenbrenner have found guinea- 

 pig serum most suitable from all standpoints, but it is important to 

 remember that the complementary activity of the serums from different 

 guinea-pigs varies, and, therefore, it is necessary to titrate each comple- 

 ment serum or hemolysin, i. e., adjust the hemolytic system, before the 

 main test is conducted. 



These quantitative factors are of great importance, and complicate 



