THE TECHNIQUE OF SERUM REACTIONS 265 



determine the quantity which will furnish a valid result. The sub- 

 stances which are used as antigens often have the power, if used in too 

 large quantity, of themselves binding complement. It is necessary, 

 therefore, to determine the largest quantity of each given antigen which 

 may be used without exerting an anti-complementary action, i.e., which 

 will not inhibit in the presence of normal serum but which will at the 

 same time inhibit hemolysis when syphilitic serum is used. This is done 

 by mixing graded quantities of the antigen with a constant 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 inac- 

 tivated hemolytic serum are added. The quantity which has given 

 complete inhibition with the syphilitic serum, but absolutely no inhibi- 

 tion with normal serum, is the one to be employed in subsequent re- 

 actions. 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 re- 

 quired. Thus if 0.05 c.c. is wanted, mix 0.5 c.c. with 9.5 c.c. salt solu- 

 tion. 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 cor- 

 puscles 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 hem- 

 olytic 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. 

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