APPLICATION OF COMPLEMENT FIXATION 197 



The antigen may be made by weighing 10 grams ox heart which has been 

 freed from blood, fat and connective tissue, ground in a meat grinder and dried 

 under a current of air from an electric fan or in a 'desiccator. It is then 

 extracted in 100 c.c. 95 per cent, ethyl alcohol, first by shaking 18 hours in an 

 electrical shaker and then standing for 5 days at 37 C. It is filtered and kept 

 tightly stoppered in an amber glass bottle in the refrigerator. For use, slowly 

 add 9.0 c.c. physiologic salt solution to i.o c.c. alcoholic extract. This constitutes 

 the "antigen dilution" of the charts. It must then be titrated to determine 

 its antilytic properties as well as its lytic powers. The following tests, of the 

 antigen may be set up after previously determining the titer of the hemolytic 

 amboceptor and complement. In the following titrations the complement is 

 diluted so that i.o c.c. contains I unit, the amboceptor so that i.o c.c. contains 

 2 units. In the first series, volume is made up by addition of salt solution, so 

 that each tube contains 4.0 c.c. and in the second series so that each tube 

 contains 2.0 c.c. 



TlTRATION OF ANTIGEN FOR ANTILYTIC PROPERTIES 



TlTRATION OF ANTIGEN FOR LYTIC PROPERTIES 



In the protocols P.H. indicates partial hemolysis, C.H. complete hemolysis 

 and ( ) no hemolysis. Thus it is seen that 0.6 c.c. is the smallest amount of 

 antigen which is antilytic and 0.4 c.c. the largest amount which is not. For 

 practical purposes one-half the latter amount, or 0.2 c.c., is the largest amount 

 which may be used. This is considerably smaller than the amount of antigen 

 which possesses hemolytic properties in itself, as shown in the second protocol. 



After obtaining this information, the antigen should be titrated to determine 

 the smallest dose that fixes complement in the presence of a known syphilitic 

 serum. A strong serum (H h++) may be obtained from a laboratory or if 

 not available a serum may be secured from 9. patient in the florid secondary 

 stage of the disease. This serum is used in constant amounts of 0.2 c.c. More 

 delicate titration is accomplished by the use of a known ++ serum either 

 alone or in addition to the H h+H~ serum. Knowing 1 that 0.2 c.c. is the largest 

 dose of antigen dilution that may be employed the test is set up with that as the 

 maximum amount of antigen, followed by decreasing doses. (See table on page 198.) 



The protocol includes the necessary controls, showing that neither antigen 

 (12), syphilitic serum (7), nor non-syphilitic serum (19) exhibits antilytic 

 powers. It shows that antigen (13), syphilitic serum (15) and non-syphilitic 

 serum produce no hemolysis. It shows that non-syphilitic human serum 

 (15-18) fails in the presence of the antigen to fix complement. It shows that 

 in the presence of syphilitic; serum the antigen solution in amounts as small as 

 o.oi c.c. fixes complement. That amount, o.oi c.c., is the fixing dose and is 

 doubled for the actual Wassermann test. 



