COMPLEMENT-FIXATION TEST IN ECHINOCOCCUS DISEASE 493 



0.5 per cent, phenol, and kept constantly at a low temperature. It is 

 highly important not to use the antigen in an anticomplementary dose, 

 hence it should be titrated before each test is made. 



Anticomplementary Titration. Dilute the fluid 1 : 10 with normal 

 saline solution, and into a series of eight small test-tubes place increasing 

 amounts as follows: 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, and 3 c.c. Add 1 c.c. of 

 complement (1 : 20) and sufficient saline solution to bring the total 

 volume in each tube up to 4 c.c. Shake gently, incubate for one hour, 

 and then add 1^ units of antisheep amboceptor (previously titrated) 

 and corpuscles. Reincubate for one to two hours; the tube showing 

 beginning inhibition of hemolysis contains the anticomplementary dose, 

 and in performing the main test, one-half to one-quarter this amount 

 may be used. 



If titration with diluted fluid does not give the anticomplementary 

 dose, the titration should be repeated with undiluted fluid. 



Alcoholic extracts of daughter cysts, the hydatid wall, or the fluid 

 have been found to give positive reactions in syphilis (Israel, Brauer, 

 Henins), and are, therefore, unsatisfactory. 



Thomsen and Magnussen speak favorably of antigen paper infil- 

 trated with cyst fluid and titrated. 



The Test. The patient's serum should be fresh, and should be heated 

 to 55 C. for half an hour. Into a series of five test-tubes place 0.025, 

 0.05, 0.1, 0.2, and 0.2 c.c. of serum. To each of the first four tubes add 

 one-half the anticomplementary dose of antigen; to all the tubes add 

 1 c.c. of complement (1 : 20) and sufficient saline solution to bring the 

 total volume up to 3 c.c. 



The fifth tube is the serum control; an antigen, hemolytic system, 

 and corpuscle control should be included, as is usual in complement- 

 fixation tests. A normal serum may be included, and, if possible, a 

 known positive serum. 



After incubating for an hour at 37 C., add 1J^ units of hemolytic 

 amboceptor and 1 c.c. of the corpuscle suspension to each tube. Re- 

 incubate for an hour or longer, depending upon the hemolysis of the 

 controls. Marked or complete inhibition of hemolysis in the fourth 

 tube (0.2 c.c. patient's serum), with lesser degrees of inhibition in the 

 other tubes of the series, indicates a positive reaction. Larger doses of 

 patient's serum should not be used on account of the probability of non- 

 specific complement fixation. 



