GENERAL TECHNIC 455 



As a general rule, the hemolytic titration is not absolutely necessary. 

 It may be conducted with the anticomplementary titration by adding 

 another tube or two to the foregoing series, with higher doses of extract; 

 or this titration may be conducted separately, and without complement 

 and hemolysin, by using the same doses of antigen with 1 c.c. of cor- 

 puscle suspension and sufficient salt solution to bring the total volume in 

 each tube up to 3 or 4 c.c. 



3. Antigenic Titration. As previously stated, this titration is not 

 absolutely necessary, as one-fourth the anticomplementary dose of an 

 extract may be used in the main test. For instance, in the foregoing 

 titration 0.3 or 0.4 c.c. may safely be used in making the test for the 

 syphilitic reaction. Different extracts vary, however, in their anti- 

 genie value. Some may be highly anticomplementary and have a 

 comparatively low antigenic value; purer extracts, such as acetone- 

 insoluble lipoids or cholesterinized alcoholic extracts of heart, are 

 largely free from anticomplementary action, and at the same time 

 possess a high antigenic value. It is advisable, therefore, to use an 

 antigen whose full antigenic as well as anticomplementary doses are 

 known, for, while it is necessary to use sufficient antigen, it is not advisable 

 to use a larger amount than is necessary. 



For this titration all antigens except alcoholic extracts of syphilitic 

 liver, should be diluted 1 : 20 with normal salt solution. Usually the 

 antigenic unit is so much lower than the anticomplementary unit that 

 it is best determined with a more dilute antigen. 



The titration is conducted in a manner similar to the anticomple- 

 mentary titration, except that 0.2 c.c. of fresh and inactivated serum 

 from a known and untreated syphilitic person is added to each tube. 

 Increasing doses of antigen, patient's serum, and complement are 

 mixed, shaken, and incubated for one hour. Two units of hemolysin 

 and corpuscles are then added, the tubes shaken and incubated for 

 another hour, after which the preliminary reading is made. The final 

 reading is taken after the tubes have been placed over night in a refriger- 

 ator at low temperature. That amount of antigen that shows just complete 

 inhibition of hemolysis is taken as the antigenic unit (Fig. 112). In con- 

 ducting the syphilis reaction two to four times this unit is used, providing 

 that these amounts are at least four or five times less than the anticomple- 

 mentary dose. This larger antigenic dose is advisable, because the exact 

 unit may not be sufficient with serums containing but small amounts 

 of syphilis antibody such as those of treated or long-standing cases of 

 lues. 



