288 INFECTION AND IMMUNITY. 



of 95 per cent, alcohol for a week at 37 C. This 

 extract is filtered and evaporated by means of a 

 fan at a temperature below 40 C. The residue 

 is extracted with ether and the ether evaporated. 

 This residue is again taken up in ether and frac- 

 tionated twice with acetone to remove the acetone- 

 soluble hemolytic substances. The acetone-insol- 

 uble residue is evaporated to dryness and extracted 

 with 95 per cent, alcohol. This solution is used 

 as a stock antigen and suspensions in salt solution 

 are used as material for tests. 



A titration of antigen should then be carried 

 out as follows: A serum from a known case of 

 syphilis should be obtained and 0.1 c.c. of the 

 inactivated serum added to each of a series of 

 tubes. Another series of controls is made by the 

 use of a like quantity of inactivated normal serum. 

 To each of the tubes of the test series graded 

 varying amounts of antigen are added and like 

 amounts added to the control tubes. Complement 

 0.1 c.c. is added to each of all of the tubes and 

 the set incubated one hour. At the end of this 

 time, two units of amboceptor for sheep's cor- 

 puscles and 1 c.c. of 5 per cent, suspension of 

 sheep's corpuscles are added and the tubes incu- 

 bated for another hour. The smallest amount of 

 antigen which, with the syphilitic serum, will bind 

 complement, is indicated by the tube containing 

 the lowest quantity of antigen in which hemolysis 

 has not taken place. The control tube containing 

 a like quantity of antigen but with normal serum 

 should be completely hemolyzed. 



Having now standardized both antigen and 

 hemolytic amboceptor, the test of the serum in 



