SEROLOGICAL METHODS 



221 



After the primary incubation is complete, 1 .0 ml of sensitized erythro- 

 cytes is added to each tube. The secondary incubation is conducted at 

 37°C for 15-30 min. 



When the results are read, absence of hemolysis indicates fixation of 

 complement due to union between the antigen and antiserum. Partial 

 hemolysis represents a weaker reaction, and complete hemolysis is a nega- 



tive reaction. The control tubes 8, 9, and 10 should display complete 

 hemolysis, while the erythrocyte control, tube 11, should not be lysed. 

 Failure to observe lysis in tube 8 indicates that the antiserum was anti- 

 complementary at this dilution. If this occurs, another test should be 

 run, using all dilutions of antiserum but substituting saline for the 

 antigen. This will show if the anticomplementary range extends far 

 enough to obscure the titer of the serum. Tube 9 is a control on the 

 antigen, and this control is usually satisfactory if the preliminary titra- 

 tions on the antigen have been performed correctly; however, some anti- 

 gens may become anticomplementary after storage for some time. 



In addition to the controls described, it is advisable to include a titra- 

 tion with known positive and known negative serum when a new system is 

 being studied. The negative (preimmunization) serum titration is par- 

 ticularly important, since some antigens may react nonspecifically with 

 normal serum. 



BIBLIOGRAPHY 



Boyd, W. C. 1947. "Fundamentals of Immunology." Interscience Publishers, 



Inc., New York. 

 Bukantz, S. C, C. R. Rein, and J. F. Kent. 1946. Studies in complement fixation. 



II. Preservation of sheep's blood in citrate dextrose mixtures (modified Alsever's 



