194 



TECHNIQUE OF THE COMPLEMENT FIXATION METHOD 



1 . An excess of antiserum can interfere with the fixation of complement. 



2. The antiserum if used in large quantities can bind complement of its 

 own accord, without the addition of the human serum. It is therefore 

 best to ascertain by titration the smallest quantities of antiserum which 

 may satisfactorily be employed, as the complement fixation test must 

 be sufficiently delicate to determine o.oooi c.cm. of the human serum. 



Diminishing amounts of antiserum are mixed with o.oooi c.cm. of 

 human serum and o.i c.cm. of complement. A control series is made 

 wherein the human serum is replaced by the same amounts of saline. 

 (The quantity in all tubes should be made uniform by the addition of 

 normal salt solution, but the total amount of fluid in each tube should not 

 exceed 2.3 to 2.5 c.cm.) The tubes are incubated for i hour and the 

 hemolytic amboceptor and red blood cells added. After two hours at 

 37 C. the results are read. The o.oooi c.cm. of the serum is added in 

 the form of 0.2 c.cm. of a i 12000 dilution. 



TABLE III. 



The antiserum itself as seen in the control series (B) does not exhibit 

 any tendency to interfere with hemolysis, even in the amount of o.i c.cm. 

 (larger quantities never come into consideration). On the other hand, 

 series (A) shows that the larger amounts of the antiserum do not bind 

 complement as thoroughly as the medium doses. The zone of complete 

 complement fixation lies between 0.05 and 0.025 c.cm. of the antiserum. 

 It is advisable as a general rule to choose about one and one-half to two 

 times this minimum quantity. Thus from Table III it can be noted that 

 0.2 c.cm. of a i :6 dilution could well be adopted as a test dose for com- 



