532 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



incubating for an hour at 37 C., amboceptor and corpuscles are added 

 to test for free complement. An absence of hemolysis indicates that 

 complement has been fixed by the antiserum and soluble typhoid antigen 

 in the serum of the patient. As a rule, and for purposes of diagnosis, 

 this order of procedure is reversed: the antigen is furnished and then 

 sought for in the patient's serum (p. 498), as in the gonococcus fixation 

 test, syphilis reaction, etc. 



An immune serum is prepared by immunizing rabbits with increasing 

 doses of an emulsion of the bacteria which we wish to test for in the 

 patient's serum. 



The bacterial extract is prepared as follows: Make cultures of the 

 bacteria on slants of agar; wash off a sufficient number with normal 

 saline solution until 20 or 30 c.c. of a heavy emulsion are secured; add 

 0.4 per cent, of phenol, and shake mechanically with glass beads for 

 twenty-four hours; then heat to 60 C. for an hour, and either centri- 

 fuge thoroughly or filter through a Berkefeld filter. The clear filtrate 

 should be preserved in a tightly stoppered bottle in an ice-chest. It is 

 well to titrate this extract for its anticomplementary dose. As a rule, 

 these extracts are free from anticomplementary action until relatively 

 large doses are employed. 



The antiserum is heated to 55 C. for half an hour and titrated with 

 0.01 c.c. of the bacterial extract (0.1 c.c. of a 1: 10 dilution) in 10 doses, 

 ranging from 0.1 c.c. to 1.0 c.c. Double the dose giving complete fixa- 

 tion of complement is used in testing for the bacterial antigen in human 

 serum. 



In conducting this test the patient's serum is heated to 55 C. for 

 half an hour, and decreasing doses, ranging from 0.5 c.c. to 0.01 c.c., 

 are placed in a series of test-tubes together with double the titrated dose 

 of antiserum. Complement and salt solution are now added, and after 

 incubating for an hour at 37 C., amboceptor and corpuscles are added 

 and the tubes reincubated. The general technic and controls are the 

 same as those previously described. 



The test has some value in special research work, but for practical 

 use it has given way to the agglutination reactions and complement- 

 fixation tests for the detection of antibody with a known antigen. 



COMPLEMENT-FIXATION TEST IN CANCER 



None of the various complement-fixation methods that have been 

 advocated from time to time in the diagnosis of cancer have proved of 



