436 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



dilution; I have found it much more accurate, simple, and economical, 

 however, to prepare one stock dilution, which is titrated with each 

 complement and corpuscle suspension before each day's work. For 

 example, if a serum is known to have a titer of 1 : 2000, 'an ampule 

 (0.5 c.c. of serum) is diluted with 200 c.c. of salt solution; this gives a 

 dilution of serum approximately 1:400, of which 0.2 represents one 

 hemolytic dose. The titration must be repeated each time to make 

 sure of this, because the complement of different pigs may vary in 

 activity, and the chief object is to adjust the amboceptor and com- 

 plement to each other. 



Titration of Amboceptor. Into a series of six test-tubes place in- 

 creasing amounts of the amboceptor dilution: 0.05 c.c., 0.1 c.c., 0.15 

 c.c., 0.2 c.c., 0.25 c.c., and 0.3 c.c. Add 1 c.c. of complement (1 : 20) 

 and 1 c.c. of corpuscle suspension to each tube, and sufficient salt 

 solution to make the total volume in each tube about 4 c.c. Shake 

 gently and incubate for one hour at 37 C. At the end of this time 

 the tube showing just complete hemolysis contains one hemolytic 

 dose, or unit of amboceptor. In the tests double this amount, or two 

 units, is used. 



The amboceptor titration is very important. Under no cir- 

 cumstances should the same dose be used day after day without 

 titration, because the complement of different guinea-pigs may vary 

 in its activity, and these variations would be detected. and would be 

 adjusted in this titration. For example, with a weaker complement 

 the dose of amboceptor required to effect complete hemolysis becomes 

 higher; each new corpuscle suspension may also vary slightly in the 

 actual number of cells contained in 1 c.c., but this makes no difference 

 when each suspension is titrated with the complement and ambo- 

 ceptor to be used in the day's work. This titration is set up first, 

 and while it is in the incubator, the main tests are arranged. 



4. Antigen. Alcoholic extract of syphilitic liver or acetone- 

 insoluble lipoids of proved value may be used. It is well to estimate 

 just how much antigen will be required for the tests on hand, so that 

 no waste will occur, as fresh emulsions are better than old ones carried 

 over from day to day. The dose should be at least double the titrated 

 antigenic unit, or one-fourth of the anticomplementary dose. For 

 instance, if an alcoholic extract of syphilitic liver diluted 1 : 10 is 

 found on titration to be perfectly antigenic in doses of 0.2 c.c., and 

 not anticomplementary in amounts under 2 c.c., then 0.4 c.c. maybe 

 used in making the tests, as this amount is still about five times less 



