120 THE PRINCIPLES OF IMMUNOLOGY 



Quantitative Relations of Amboceptor and Antigen. By subse- 

 quent studies of different mixtures, it was found that the unit of 

 standard corpuscle suspension can take up considerably more than one 

 unit of amboceptor. This amount varies with the total quantity of im- 

 mune body present. For example, Muir found that on addition of 

 twelve doses of amboceptor one dose remained free, on addition of six- 

 teen doses of amboceptor two doses remained free, on addition of 

 twenty doses three doses remained free and on the addition of twenty- 

 three doses of amboceptor, four doses remained free. When, how- 

 ever, the mixture of complement, amboceptor and red blood- 

 corpuscles is properly adjusted, the reaction completely uses up the 

 amboceptor, complement and, by hemolysis, all the red blood-corpuscles. 

 Correspondingly, if two units of complement are employed in the 

 presence of one unit of amboceptor, it does not follow that after 

 the reaction one unit of complement will remain free. As a matter 

 of fact, practically the entire two units of complement will be utilized 

 in the reaction. Nevertheless, increasing the amounts of complement 

 will leave more and more complement free in the supernatant fluid. 

 These points will be made somewhat clearer after subsequent experi- 

 ments have been outlined. 



Relative Affinities of Amboceptor and Complement. In the in- 

 troductory paragraph it was pointed out that the amboceptor has a 

 special affinity for the antigenic red blood-corpuscles, but that the com- 

 plement has no such affinity. This is illustrated by the fact that when 

 red blood-corpuscles are set up against amboceptor they will absorb 

 the amboceptor, but if they are set up in the presence of complement 

 they will not absorb complement. 



The following experiment illustrates this point: Two centrifuge tubes 

 are marked A and B. In tube A are placed i.o c.c. standard erythrocyte sus- 

 pension (5 per cent, suspension) and i.o c.c. inactivated immune serum so 

 diluted as to contain two units amboceptor. This tube is incubated at 37 C. 

 for thirty minutes and then centrifuged. The supernatant fluid is pipetted 

 into a tube marked A 2. The erythrocyte sediment in tube A is washed in 

 salt solution, again centrifuged and the supernatant fluid discarded. The 

 sediment in tube A is resuspended in i.o c.c. salt solution and two units of 

 complement, i.e., i.o c.c. i-io dilution are added. To tube A 2 are added two 

 units complement (i.o c.c. i-io dilution) and i.o c.c. 5 per cent, 'erythrocyte 

 suspension. These tubes are incubated for one hour at 37 C. Tube A will 

 show hemolysis because the sedimented corpuscles have absorbed ambo- 

 ceptor and the addition of complement is sufficient to complete the reaction. 

 Tube A 2 will not show hemolysis because the amboceptor is not in the 

 supernatant fluid and the complement is not sufficient to lake the added 

 corpuscles. At the same time the converse of the foregoing experiment may 

 be conducted. In tube B are placed i.o c.c. standard erythrocyte suspension 

 (5 per cent, suspension) and i.o c.c. fresh guinea-pig serum diluted i-io. 

 This is incubated thirty minutes at 37 C, centrifuged and the sediment 

 washed. The supernatant fluid is placed in tube B 2. The sediment in tube B 

 is resuspended in i.o c.c. salt solution and i.o c.c. immune serum so diluted 

 as to contain two units amboceptor is added. To the supernatant fluid in 

 tube B 2 are added i.o c.c. immune serum (two units amboceptor) and i.o c.c. 

 erythrocyte suspension. These tubes are incubated for one hour at 37 C. 

 Tube B 2 will show hemolysis because the supernatant fluid after the centrifu- 

 gation still contains complement, so that the addition of amboceptor and 

 erytnrocytes permits of completion of the reaction. Tube B will not show 



