172 



THE TECHNIQUE OF COMPLEMENT FIXATION. 



own accord; without the addition of the human serum, for example. 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.c. of the human serum. 



Diminishing amounts of antiserum are mixed with .0001 c.c. of human 

 serum and o.i c.c. 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.c.). 

 The tubes are incubated for i hour and the hemolytic amboceptor and red 

 blood cells added. After two hours at 37 the results are read off. The 

 .0001 c.c. of the serum is added in the form of 0.2 c.c. of a i : 2000 dilution. 



TABLE III. 



Amounts of antiserum 

 in cubic-centimeters. 



Series A contains antiserum Series B (control) contains anti- 



+ o.oooi c.c. human serum serum + 0.2 c.c. physiological 



(1:2000.02)4-0.1 guinea-pig's saline + o. i of guinea-pig's serum 

 serum. 



One hour at 37. 

 + 0.001 c.c. of amboceptor +i 

 c.c. of 5 per cent, ox's-blood. 



One hour at 37. 



+ 0.001 c.c. of amboceptor + i c.c. 

 5 per cent, of ox's-blood. 



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

 amount of o.i c.c. (larger quantities never come into consideration) exhibit 

 any tendency to interfere with hemolysis. 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.c. 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.c. of a i : 6 dilution 

 would be well adopted as a test dose for complement fixation. If it is 

 required to know how delicate the complement fixation reaction can be with 

 this dose of antiserum, the following experiment (Table IV) is undertaken : 



Diminishing amounts of human serum are mixed with a constant quan- 

 tity of complement and with the constant test dose of antiserum. At the 



