PRINCIPLES OF COMPLEMENT FIXATION WITH BACTERIAL ANTIGENS 501 



ment. After an hour the proper dose of hemolytic amboceptor and cor- 

 puscles is added. The readings may be made an hour or two later, or 

 after the tubes have been allowed to settle in a refrigerator. That tube 

 showing just complete inhibition of hemolysis contains the antigenic unit. 

 For the main test it is well to use double this amount, providing this 

 dose is not more, and preferably less, than half the anticomplementary 

 unit. 



The antigenic titration is not always satisfactory, for when an arti- 

 ficial immune serum is used the concentration of antibodies may yield 

 a much stronger reaction than one would expect in testing human serums. 

 Further than this, the antibody content in antiserums varies considerably 

 so that the antigenic unit fluctuates according to the particular serum 

 used in making the titration. In general, therefore, it is sufficient to 

 determine the anticomplementary unit and to use half or quarter this 

 amount in performing the main test. 



After antigens are prepared they may require further dilution with 

 saline solution. This can be determined only by experience and as the 

 result of a trial titration. 



As watery extracts are prone to deteriorate, it should be made a rule 

 that the anticomplementary dose be determined each time before the main 

 test is conducted. 



It has quite generally been proved that alcoholic extracts of bacteria 

 do not yield satisfactory antigens, despite the advantage to be gained 

 because of their stability. 



Principles of Complement Fixation with Bacterial Antigens. The 

 principles of complement fixation in general should be thoroughly under- 

 stood. 



After making considerable comparative studies with various methods, 

 I am convinced that, in the final analysis, a simple technic is best. I 

 titrate the complement or use a relatively small but safe dose of com- 

 plement, 1 c.c. of a 1 : 20 dilution ( = 0.05 c.c. undiluted serum), and 

 titrate the hemolytic amboceptor with this constant dose or unit of 

 complement and the corpuscle suspension. This titration is made each 

 time the reactions are performed, and with each and every complement 

 serum and corpuscle suspension. In this way differences in the activity 

 of different guinea-pig serums are readily detected and adjusted. If 

 exactly one unit of complement or amboceptor is used in conducting the 

 main test, the controls are not likely to be completely hemolyzed unless 



