CYTOLYSINS 119 



N no hemolysis, the reading being made after a period of incubation in the 

 water bath at 37 C. This period may be thirty minutes, one hour or two hours, 

 but subsequent experiments with the same system of amboceptor, complement 

 and corpuscles must be made with the same period of incubation as practised in 

 the original titration. In this laboratory one hour is the standard time for incu- 

 bation. In order to make results somewhat more clear-cut, the rack of ' test 

 tubes may be placed in the refrigerator over night and the results read the fol- 

 lowing morning. The lapse of twelve or eighteen hours time permits the cor- 

 puscles to settle to the bottom' of the test-tube ; therefore any red coloring of 

 the supernatant fluid may be interpreted as a partial or complete hemolysis, de- 

 pending on the depth of color and the amount of sediment remaining on the 

 bottom of the tube. The controls which are used in this experiment demon- 

 trate that neither complement nor inactivated amboceptor will produce hemoly- 

 sis. The result given in the above experiment indicates that at some point 

 between the dilutions 1-2500 and 1-3000 the exact end point of titration is to be 

 found. In order to determine the exact end point it is well to set up an addi- 

 tional series with dilutions of 1-2500, 1-2600, 1-2700, 1-2800, 1-2000, and 1-3000 

 with the necessary controls. If it is found that complete hemolysis takes place 

 in a dilution 1-2700 and not in the dilution 1-2800 the dilution 1,2700 is taken as 

 the end point or titer. The unit of amboceptor therefore is 1-2700 of 0.5 c.c. or 

 1-5400 of i c.c. In the experiment outlined above, the unit of amboceptor would 

 be designated as 0.5 c.c. of a 1-2700 dilution of the immune serum. 



Titration of Complement. As has been indicated previously, the amount 

 of complement in guinea-pig serum varies in different animals. Therefore sub- 

 sequent experiments with this amboceptor must be controlled by titrating the 

 complement. This may be done by setting up a series of tubes as follows, the 

 control tubes being made up to volume with salt solution : 



Erythrocytes Amboceptor Complement PP C ,,H 



suspension 1-2700 i-io 



0.5 c.c. 0.5 c.c. 0.5 c.c. CH 



0.5 c.c. 0.5 c.c. 0.4 c.c. CH 



0.5 c-c. 0.5 c.c. 0.3 c.c. PH 



0.5 c-c. 0.5 c.c. 0.2 c.c. N 



0.5 c-c. 0.5 c.c. o.i c.c. N 



0.5 c.c. ... 0.5 c.c. N 



0.5 c.c. 0.5 c.c. ... N 



0.5 c.c. ... ... N 



In this experiment it is found that 0.4 c.c. of the new complement is sufficient 

 for activating the unit of amboceptor. Therefore, whereas in the first experiment 

 0.5 c.c. i-io complement dilution was the unit of complement, in the second experi- 

 ment 0.4 c.c. i-io dilution complement is the unit. If it is found that in none of 

 these tubes complete hemolysis takes place because of weak complement, it will 

 then be necessary to set up an additional series with complement diluted 1-5 

 instead of i-io. 



Quantitative Relations of Amboceptor and Complement. The 



quantitative relationship between the amount of complement and ambo- 

 ceptor used has been very extensively studied. It is now known that 

 a larger amount of complement will require a smaller amount of ambo- 

 ceptor for the production of complete hemolysis in the standard blood- 

 corpuscle suspension and conversely a smaller amount of complement 

 requires a larger amount of amboceptor. Thus, if we use two units of 

 complement, hemolysis will occur in the presence of less than one unit 

 of amboceptor. If we use two units of amboceptor, it will require 

 less than one unit complement to produce complete hemolysis. This 

 relationship, however, is not in definite proportion. For example, if 

 four units of amboceptor are employed, one-third unit of complement 

 is necessary. This relationship is beautifully illustrated in the dia- 

 gram (Fig. 13) taken from Noguchi. 



