298 IMMUNOLOGICAL METHODS OF DIAGNOSIS 



be quite sufficient to effect a very considerable degree of hemolysis, 

 if amboceptor were present in excess. 



Some investigators, such as Noguchi, have accordingly recom- 

 mended the use of an antihuman instead of an antisheep hemolytic 

 system. I have found, however, that this is not only inconvenient, 

 but also unnecessary, as the natural antisheep amboceptor can be 

 readily removed by merely diluting the inactivated serum of the 

 patient with five times its volume of the corpuscle emulsion, and 

 incubating the mixture for thirty minutes in the water-bath at 

 37 C.; the corpuscles with the anchored " natural" antisheep ambo- 

 ceptor are then thrown down with the centrifuge, when the super- 

 natant, now already diluted, serum is ready for use. This step is 

 now carried out as a matter of routine in my own laboratories, and 

 assures perfectly satisfactory results; with the use of the Noguchi 

 antigen, and this modification the objectionable "Nachlosung" 

 (continuing hemolysis at the end of the experiment) 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 2J 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 E), 

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



1 The patient's serum has previously been freed of 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. 



