454 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



Sufficient emulsion for these titrations is prepared by diluting 0.4 

 c.c. of the alcoholic solution with 3.6 c.c. of saline solution. 



Into a series of seven small test-tubes place increasing amounts of 

 this emulsion as follows: 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.0 c.c., add 0.1 c.c. 

 (5 capillary drops) complement (40 per cent.) to each; also 1 c.c. of a 

 1 per cent, suspension of corpuscles and sufficient saline solution to 

 make the total volume in each tube about 2 c.c. Incubate at 37 C. 

 for one hour (one-half hour in water-bath), and add two units of ambo- 

 ceptor. Shake the tubes gently and reincubate for two "hours (one hour 

 on water-bath). That tube showing beginning inhibition of hemolysis 

 contains the anticomplementary dose, which should not be under 0.4 

 c.c. In the tubes containing the larger doses slight hemolysis may be 

 noticed, which is evidence of the hemolytic action of the extract. 



An eighth tube should be included, containing 0.1 c.c. diluted com- 

 plement, two units of amboceptor, and 1 c.c. of the corpuscle suspension. 

 This is the hemolytic control and should show complete hemolysis. 



2. Antigenic Titration. Since the extract is likely to have a high 

 antigenic value, it is necessary to dilute the antigen still further by plac- 

 ing 0.5 c.c. of the foregoing emulsion in a test-tube and adding 4.5 c.c. 

 of saline solution (1 : 100 dilution of the antigen). Into a series of six 

 test-tubes place increasing amounts of this emulsion as follows: 0.05, 

 0.1, 0.2, 0.3, 0.4, 0.6 c.c. To each tube add four drops (0.08 c.c.) of 

 inactivated or one drop (0.02 c.c.) of fresh active syphilitic serum; also 

 0.1 c.c. (five capillary drops) of complement (40 per cent.) and 1 c.c. of 

 1 per cent, corpuscle suspension. Then add sufficient salt solution to 

 bring the total up to 2 c.c. 



Two controls should be included: (1) The serum control, containing 

 the dose of serum, 0.1 c.c. of the complement, 1 c.c. of corpuscle sus- 

 pension, and saline solution; (2) the hemolytic control, containing at 

 this time 0.1 c.c. of complement, 1 c.c. of corpuscle suspension, and 

 sufficient saline solution. 



All tubes are incubated for one hour at 37 C. (one-half hour in 

 water-bath), after which two units of amboceptor are added to each 

 tube. The tubes are then shaken gently and reincubated for two hours 

 (one hour in water-bath). At the end of this time the two controls 

 should be completely hemolyzed, and in the series proper that tube 

 showing just complete inhibition of hemolysis contains one antigenic 

 unit. Usually the first and second tubes show some inhibition of 

 hemolysis, and in the third and other tubes hemolysis is completely 

 inhibited. In this case 0.2 c.c. of this emulsion would be one anti- 



