SEROLOGICAL METHODS 215 



COMPLEMENT-FIXATION TEST FOR MICROBIAL ANTIGENS 



In the normal serum of numerous animal species there occurs a sub- 

 stance known as alexin or complement. It is a mixture of at least four 

 fractions, chiefly globulin in nature. Although it does not increase in 

 amount following immunization, it has a role in serological reactions, 

 since it is able to combine with many antigen-antibody complexes. This 

 combination may be spoken of as the ''fixation" of complement. 



When erythrocytes are mixed with antierythrocyte serum and comple- 

 ment is added, lysis of the cells will occur. Such lysis requires both anti- 

 body and complement. Practical application is made of this lytic 

 phenomenon in the complement-fixation test where sheep erythrocytes 

 and their homologous antibody (hemolysin) are used as an indicator 

 system. 



In the complement-fixation test, two separate antigen-antibody sys- 

 tems are allowed to compete for complement. When the test is per- 

 formed the antigen and antiserum under study are mixed and a carefully 

 measured amount of complement is added. If the serum contains anti- 

 bodies that react with the antigen, complement is absorbed by the anti- 

 gen-antibody complex and no free complement remains. If, at this 

 point, sheep erythrocytes and their homologous antibody (hemolysin) are 

 added, the sheep cells will not lyse, since there is no free complement. If, 

 on the other hand, the first antigen-antibody reaction had failed to occur 

 owing to deficiency of either antibody or antigen, free complement would 

 be available and the sheep cell indicator system would lyse. 



The complement-fixation test is not necessary for some systems where 

 other antigen-antibody reactions are available; however, it sometimes 

 yields information that cannot be obtained by other procedures. The 

 test described here is a modification of the Kolmer method for syphilis 

 (Kolmer 62^ aL, 1951). 



Reagents needed for the test include 0.9 per cent sodium chloride solu- 

 tion (''saline"), a suspension of sheep erythrocytes, hemolysin, comple- 

 ment, antigen, and antiserum. 



Preparation of sheep erythrocytes. Blood is collected from the jugular 

 vein of the sheep. The erythrocytes may be preserved for several weeks 

 by mixing the blood immediately with an equal volume of sterile modified 

 Alsever solution (Bukantz et at., 1946) of the following composition: 



Glucose 20.5 g 



Sodium citrate 8.0 g 



Sodium chloride 4.2 g 



Citric acid . 55 g 



Distilled water 1 liter 



