314 IMMUNOLOGICAL METHODS OP DIAGNOSIS 



at the end of the experiment) which was formerly so frequently 

 encountered, when the older technique was employed, is no longer 

 a source of error and hence of worry. 



The test-tubes which we use measure 4 inches in length by f inch 

 inside diameter. 



METHOD. When everything is in readiness the complement and 

 amboceptor are adjusted to one another, using dilutions of 1 to 1000, 

 1 to 2000, 1 to 3000, up to 1 to 6000 of the amboceptor; 0.5 c.c. is 

 our unit of measure, and we accordingly combine 0.5 of the various 

 amboceptor dilutions with 0.5 c.c. of the complement (1 in 10) and 

 0.5 c.c. of the corpuscle emulsion (5 per cent.). The tubes are placed 

 in the water-bath at 37 C., and are frequently shaken. At the 

 expiration of thirty minutes the highest dilution is noted at which 

 complete hemolysis occurs. The amboceptor dilution to be used in 

 the actual experiment is then made 2\ to 3 times as strong. Thus, 

 if complete hemolysis occurred at 1 to 6000, we would use a 1 to 

 2500 or a 1 to 2000 dilution. 



The antigen has been previously tested, as described. With 

 human heart antigen, one can usually use a dilution of 1 to 10. 



The titers of the various reagents having thus been ascertained, 

 the experiment proper can now be carried out (tubes marked A), 

 using 0.5 c.c. of the patient's serum (1 in 5) 1 combined with 0.5 c.c. 

 of complement (1 in 10) and 0.5 c.c. of antigen (1 in 10). At the 

 same time controls (tubes marked B) are prepared in which the 

 antigen is left out, so that 0.5 c.c. of each serum is combined with 0.5 

 c.c. of complement and 0.5 c.c. of saline (in place of the antigen). The 

 A and B tubes properly marked with the patient's numbers are 

 placed in the water-bath for thirty minutes and then receive, each, 

 0.5 c.c. of the hemolytic amboceptor and 0.5 c.c. of the corpuscles. 

 They are then returned and left until the B tubes show complete 

 hemolysis, all the tubes being frequently shaken. As a general 

 rule an incubation of from ten to fifteen minutes suffices for this 

 purpose. But if need be they may be left for a longer time at any 



1 The patient's serum has previously been freed from any natural antisheep 

 amboceptors by diluting it (1 in 5) with the standard emulsion of sheep cor- 

 puscles and incubating for thirty minutes, after which the corpuscles are thrown 

 down by centrifugation, when the supernatant fluid is pipetted off and can be 

 immediately used in the experiment. 



