SEROLOGICAL METHODS 



219 



cient combining power, and these properties must be measured when a 

 new antigen is prepared. 



Anticomplementary activity of antigen. The stock antigen is diluted in 

 tenfold (1 : 10, 1 : 100, 1 : 1,000, etc.) steps for a rough titration or in twofold 

 increments after the approximate range has been established. Then to 

 each of a series of 15- by 85-mm tubes are added 1.0 ml of complement 

 (two full units), 0.5 ml of saline solution, and 0.5 ml of a given antigen 

 dilution. These are shaken and then placed at 37°C for 1 hr. At this 

 point 1.0 ml of sensitized erythrocytes is added and the tubes are shaken 

 and placed once again in the 37° water bath for 30 min. Hemolysis 

 should occur, and any evidence of inhibition shows anticomplementary 

 activity of that particular antigen dilution. 



Hemolytic activity of antigen. Into a series of test tubes, place 0.5-ml 

 portions of the antigen dilutions used in the foregoing section. Add 

 2.0 ml of saline and 0.5 ml of nonsensitized 2 per cent erythrocyte suspen- 

 sion to each tube. Mix, and incubate in a 37° bath for 1 hr. If hemol- 

 ysis is observed, the antigen concentration used in the actual test must 

 fall below the hemolytic level. 



Antigen titration. In arriving at the correct amount of antigen to use 

 in the complement-fixation test, one may choose a fixed concentration of 

 antibody and titrate various dilutions of antigen against it. It occa- 

 sionally happens, however, that such a procedure yields misleading 

 results, and it is generally safer to titrate both dilutions of antibody and 

 dilutions of antigen in a ''checkerboard" protocol of the type shown 

 under ''Complement Fixation" in the chapter on viruses or Table 22 in 

 this chapter. 



One should choose an antiserum known to contain a reasonably high 

 titer of antibody. Serial twofold dilutions of the serum (1:2, 1:4, 1:8, 

 etc.) are placed in 15- by 85-mm test tubes, using 0.5 ml per tube. At the 

 same time, serial twofold dilutions of antigen are prepared and added to 

 the serum dilutions ; thus, if five dilutions of serum and eight dilutions of 

 antigen are being used, 40 tubes are required. It is also customary to 

 include controls as shown in Table 24. The concentrations of antigen 

 used must be less than the hemolytic and anticomplementary levels. 



Saline and complement are added to the tubes (Table 24), and the 

 primary incubation is made at 37°C for 1 hr. 



After the primary incubation, sensitized erythrocytes are added and a 

 secondary incubation is conducted in the 37° bath for 30 min. The tubes 

 are read as described for the complete test. The optimal amount of 

 antigen is the dilution that gives the most sensitive reaction with the 

 smallest concentration (highest dilution) of antibody. 



Antiserum. The antiserum is prepared by immunizing rabbits oi 

 other experimental animals as described earlier. The serum should be 



