512 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



The anticomplementary dose is then determined by titration. The 

 antigen is diluted 1 : 20 by mixing 1 c.c. with 19 c.c. of normal saline 

 solution. To a series of seven test-tubes add increasing amounts of 

 diluted antigen as follows: 0.1, 0.2, 0.4, 0.6, 0.8, 1, and 2 c.c. Add 1 c.c. 

 of complement serum (1 : 20) to each tube, and sufficient salt solution 

 to bring the total volume in each up to 3 c.c. Incubate for an hour at 

 37 C., and add 2 units of antisheep amboceptor titrated just previous 

 to making the test (see p. 399) and 1 c.c. of sheep corpuscle suspension. 

 Reincubate for an hour or an hour and a half. At the end of this time 

 that tube showing beginning inhibition of hemolysis contains the anticom- 

 plementary unit, and one-fourth to one-half this amount is used in making 

 the main test. 



An antigenic titration may also be made, but this is not absolutely 

 necessary. To a series of tubes containing 0.05, 0.1, 0.15, 0.2, 0.25, and 

 0.3 c.c. of diluted antigen add 0.1 c.c. of fresh heated glanders serum 

 (known to yield a positive reaction) and 1 c.c. of complement serum 

 (1 : 20) and sufficient salt solution. Incubate for one hour and add 2 

 units of hemolytic amboceptor and corpuscles. After a second incuba- 

 tion of from one to two hours that tube showing just complete inhibition 

 of hemolysis contains the antigenic unit, and double this amount may be 

 used in performing the main test, providing that it is still one-half or, 

 better, but one-quarter the anticomplementary unit. 



A hemolytic system control is included in both titrations, and in the 

 antigenic titration an additional control on the serum, to determine 

 whether it is free from anticomplementary action. 



The dilution here advised may be too low; if this is the case, the 

 titrations must be repeated with the antigen diluted 1 : 50 or 1 : 100. 



The Test. The external jugular vein of the horse is punctured with 

 a sterile needle, and from 5 to 10 c.c. of blood is collected in a centrifuge 

 tube or other glass container, which preferably should be sterile. The 

 clear serum is heated to 55 C. for one-half hour just before the tests are 

 conducted. 



Into a series of four small test-tubes place the following doses of 

 serum: 0.05, 0.1, 0.2, and 0.2 c.c. To the first three tubes add the 

 proper dose of antigen; to all tubes add 1 c.c. of complement (1 : 20) and 

 sufficient salt solution to bring the total volume up to 3 c.c. 



The following controls should be included: 



1. The serum control on each serum is conducted in the fourth tube 

 of each set. 



2. A known positive serum should be tested in the same manner. 



