CYTOLYSINS 121 



hemolysis because the corpuscles in the sediment have not taken up any 

 complement, and the addition of amboceptor is not sufficient for the 

 reaction to occur. 



Selective Absorption of Amboceptor and Complement. Not only 

 is it possible to show, as has been done in the preceding experiment, 

 that red blood-corpuscles will combine with amboceptor and not with 

 complement, but if conditions are so arranged that hemolysis is pre- 

 vented, it is possible to demonstrate that red blood-corpuscles will 

 selectively absorb amboceptor from a mixture of amboceptor and 

 complement. In order to prevent hemolysis, it is necessary to permit 

 the absorption to take place at o C. Not only must this precaution 

 be observed, but the tubes must be cooled, the various reagents in the 

 mixture must be cooled in advance and the centrifuge carrier must 

 also be cooled. 



The various reagents are placed in test tubes and all the tubes placed in 

 a mixture of salt and ice. Into a cold centrifuge tube are placed i.p c.c. 

 5 per cent, erythrocyte suspension, i.o c.c. inactive immune serum so diluted 

 as to contain two units amboceptor and i.o c.c. guinea-pig serum, i-io dilu- 

 tion. This tube remains in the salt-ice mixture for thirty minutes and is 

 then centrifuged. The supernatant fluid is poured off and divided so that 

 one-half the amount is placed in each of two tubes. The sediment is washed 

 in cold salt solution and resuspended in 4.0 c.c. cold salt solution. These 

 4.0 c.c. are divided between two tubes. The four tubes so prepared are set 

 up as follows: 



TUBE i 



Supernatant fluid 1.5 c.c. 



Fresh guinea-pig serum, i-io 0.5 c.c. 



5 per cent, erythrocyte suspension 0.5 c.c. 



TUBE 2. 



Supernatant fluid 1.5 c.c. 



Immune rabbit serum 0.5 c.c. 



5 per cent, erythrocyte suspension 0.5 c.c. 



TUBE 3. 



Sediment 2.0 c.c. 



Fresh guinea-pig serum, i-io 0.5 c.c. 



TUBE 4. 



Sediment 2.0 c.c. 



Immune rabbit serum 0.5 c.c. 



These tubes are incubated for one hour at 37 C. Inasmuch as the 

 supernatant fluid no longer contains amboceptor, tube i will fail to show 

 hemolysis, but in the case of tube 2 the amboceptor is added, and since the 

 supernatant fluid contains complement which has not been absorbed by the 

 corpuscles, hemolysis will result. The sediment has absorbed amboceptor 

 from the mixture ; therefore, in the case of tube 3, the addition of fresh guinea- 

 pig serum will serve to produce hemolysis. The sediment has not, however, 

 taken up any complement, the addition of the immune serum in tube 4 will 

 not serve to complete the reaction, and hemolysis will not occur. By the 

 use of sera containing other hemolytic amboceptors, it is possible to show 

 not only that absorption of amboceptor may occur from a complement 

 amboceptor mixture, but that this absorption is specific for the particular 

 amboceptor concerned. 



Influence of Amount of Complement. Although, as will be shown 

 subsequently, the concentration of complement plays a part in the com- 



