THE TECHNIQUE OF SERUM REACTIONS 269 



mulation of the Wassermann reaction a great many modifications have 

 been suggested by various workers, some of them being radical changes 

 involving the altering of the hemolytic system; others, however, merely 

 adding precautions here and there to increase the delicacy of the reac- 

 tion. The literature on this subject is too voluminous to be com- 

 pletely covered. Wejndicate, therefore, some of the most important 

 changes from the original that have been found valuable, and give in 

 greater detail the methods as -at present .in use in our own laboratory. 



Bauer has called attention that human serum contains a certain amount of 

 natural- hemolysin for sheep corpuscles. In his original modification, therefore, he 

 does not use hemolytic rabbit serum as amboceptor. His modification as a whole 

 cannot be accepted for general use because human sera do not contain a uniform 

 amount of hemolysin for sheep cells, and some contain none whatever. However, 

 the presence of natural amboceptor, so-called, in human sera is taken account of 

 by many workers, and it is important to recognize this, since naturally it adds to 

 the amboceptor added with sensitized cells -and leads to a lack of uniformity in 

 the dosage of amboceptor in individual tubes if included. 



Noguchi has worked out a test in which the difficulties presented by the pres- 

 ence of normal sheep amboceptor are eliminated, in that he uses an antihuman 

 hemolytic serum and human cells as the hemolytic system. It enables him also to 

 use the cells of the patient or of any other human being, thus eliminating the neces- 

 sity of getting fresh sheep cells. His tests are set up as follows: 



drop patient's serum + complement (0.1 c.c. of 40 per cent guinea-pig serum) + antigen- 



drop patient's serum + complement. (No antigen.) 

 drop known syphilitic serum + complement + antigen, 

 drop known syphilitic serum -f- complement. (No antigen.) 

 drop known normal serum + complement + antigen, 

 drop known normal serum + complement. (No antigen.) 



Tube 1. 

 Tube 2. 

 Tube 3. 

 Tube 4. 

 Tube 5. 

 Tube 6. 

 Tube 7. Complement alone (for hemolytic system control). 



To each tube then add 1.0 c.c. of the one per cent emulsion of human corpuscles. 

 Shake mixtures thoroughly and incubate or place in water bath at 38-40 C. for one 

 hour. Then add to each tube 2 units of antihuman amboceptor (serum of rabbit 

 immunized with human cells) and replace in water bath for one hour. At the end 

 of this time in a positive test there will be no hemolysis in Tubes 1 and 3 while all 

 the other tubes will show hemolysis. 



The method of Noguchi is still used by a few investigators, but is 

 not at present in common use, though we have no doubt that if sys- 

 tematically followed it would develop as quite satisfactory. 



The tests are done in our own laboratory with the original sheep 

 cell antisheep serum hemolytic system. They are done in half quan- 

 tities, titrations being made with 0.5 c.c. of a 5 per cent emulsion of 

 washed sheep cells. Each day the complement (fresh guinea-pig diluted 

 1 : 10) is titrated with cells sensitized with 2 units of stock amboceptor. 

 Fresh amboceptors are titrated from time to, time so that a reasonable 

 constancy is obtained. The hemolytic system is kept as constant as 

 possible from day to day. The unit (minimal hemolytic amount) of 

 a new specimen of amboceptor is determined by titrating it on a num- 

 ber of successive days with 0.5 c.c. of 5 per cent cells and 0.5 c.c. of 

 10 per cent guinea-pig sera, readings being made at the end of a half 



