472 



THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



tion must be performed with every complement serum before the main 

 tests are conducted. In order to allow for the anticomplementary 

 action of the antigen the complement is titrated as suggested by Thorn- 

 sen, in the presence of the dose of antigen, as determined by titration, 

 to be used in the main tests, as follows: In each of a series of six test- 

 tubes place the dose of antigen (as, for example, 0.1 c.c. of 1 : 20 dilution 

 of acetone insoluble lipoids) ; add the following amounts of complement 

 1 : 10 with an accurate pipet: 0.1 c.c., 0.2 c.c., 0.3 c.c., 0.4 c.c., 0.5 c.c., 

 and 0.6 c.c. Add sufficient salt solution to make the total volume in 

 each tube about 2 c.c., mix gently and incubate in a water-bath at 38 C. 

 for one hour, then one unit of hemolysin and 0.5 c.c. of 2| per cent, 

 suspension of cells are added, the tubes shaken and reincubated in the 

 water-bath for an hour, when the reading is made. The unit of comple- 

 ment is the smallest amount producing complete hemolysis. The results 

 of a titration are shown in Table 15a and Fig. 110. 



TABLE 15a. RESULTS OF COMPLEMENT TITRATION IN THE PRES- 

 ENCE OF ANTIGEN 



4. Antigen. A large number of comparative tests with the same 

 sera and a variety of antigens have shown that a good extract of acetone- 

 insoluble lipoids is best adapted for this reaction. As a general rule these 

 extracts possess a marked degree of antigenic sensitiveness and are us- 

 ually free of anticomplementary action except in very large doses. Each 

 extract must be titrated to determine the proper dose to employ; as a 

 general rule it is sufficient to titrate an extract once every two months 

 providing it is carefully refrigerated and is yielding satisfactory results 

 in complement-fixation tests. 



In conducting these titrations a mixed complement serum diluted 

 1 : 10 is titrated in the following doses with one unit of hemolysin and 



