THE TECHNIQUE OF SERUM REACTIONS 319 



This is done by mixing graded quantities of the antigen with a con- 

 stant quantity of complement (0.1 c.c. of fresh guinea-pig serum), in 

 duplicate sets, adding to each tube of one set 0.2 c.c. of a normal 

 serum, and to the other 0.2 c.c. of a known syphilitic serum. These 

 substances are allowed to remain together for one hour and then red 

 blood corpuscles and inactivated hemolytic serum are added. The 

 quantity which has given complete inhibition with the syphilitic serum, 

 but absolutely no inhibition with normal serum, is the one to be 

 employed in subsequent reactions. Before actual use, it is convenient 

 to make a dilution of antigen in salt solution in such a way that 1 c.c. 

 shall contain the amount required. Thus if 0.05 c.c. is wanted, mix 

 0.5 c.c. with 9.5 c.c. salt solution. Then 1 c.c. of this can be added to 

 each tube in the test. 



II. The Hemolytic Serum. The hemolytic amboceptor, for the 

 reaction, is obtained by injecting into rabbits the washed red blood 

 corpuscles of a sheep. A 5 per cent emulsion of the corpuscles is made 

 and of this 5 c.c., 10 c.c., 15 c.c., etc., are injected at intervals of five 

 or six days. Three or four graded injections of this kind are usually 

 sufficient to furnish a serum of adequate hemolytic power. The injec- 

 tions may be made intraperitoneally or intravenously. About nine or 

 ten days after the last injection of corpuscles, the rabbit is bled from 

 the carotid artery and the serum obtained by pipetting it from the 

 clot. 



It is best to have a hemolytic serum of high potency in order that 

 the quantities used for the reaction may be as small as possible. This 

 is desirable because of the fact that the serum may contain small 

 amounts of precipitins for sheep's serum, due to insufficient washing 

 of the corpuscles employed in the immunization. 



It is necessary to carefully titrate the hemolytic serum. For the 

 actual reaction most observers make use of two hemolytic units. A 

 hemolytic unit is the quantity of inactivated immune serum which, in 

 the presence of complement, suffices to cause complete hemolysis in 

 1 c.c. of a 5 per cent emulsion of washed blood corpuscles. It is the 

 custom in most laboratories today to halve all the quantities, using 

 0.5 c.c. of the suspension instead of 1.0 c.c. and other ingredients 

 accordingly. Noguchi 15 has pointed out very clearly the dangers of 

 not delicately adjusting the quantity of amboceptor used in the reac- 



NogucJii, Poc. Soc. for Exper. Biol. and Med., VI, 3, 1909. 



