308 IMMUNOLOGICAL METHODS OF DIAGNOSIS 



As the antigen in itself is capable of absorbing a certain amount 

 of complement it will be found that with the stronger emulsions 

 either no hemolysis at all occurs, or partial hemolysis only takes 

 place. For the actual experiment two-thirds of that strength is 

 chosen which first gives complete hemolysis. In the above example 

 it will be noted that this was first obtained with the 1.5 in 10 dilu- 

 tion; in this case then we would use the antigen in a dilution of 1 

 in 10, and this would represent its titer. After this has been ascer- 

 tained the antigen (in the dilution just determined) is next tested 

 against a known syphilitic serum and a known normal serum, both 

 having been inactivated by heating for thirty minutes in the water- 

 bath at 56 C., and extracted with sheep corpuscles, as described 

 below (sub. 5), 0.5 c.c. of the diluted serum being substituted for 

 the 0.5 c.c. measure of saline, corresponding to the second column 

 in the table above. With the known syphilitic serum no hemolysis 

 should then result, while with the normal serum hemolysis must be 

 complete at the expiration of thirty minutes in the water-bath. 

 Occasionally it happens that a considerable degree of fixation of 

 complement occurs with normal sera even. Such antigens are 

 evidently not fit for use, for although every serum possesses anti- 

 complementary properties to a certain extent, it would be danger- 

 ous to let the boundaries of the normal and the abnormal overlap. 

 In testing out the antigen it is further well to set the tube, in which 

 complete fixation was noted at the expiration of thirty minutes, aside 

 in the ice-box for a few hours and to examine it from time to time to 

 ascertain whether hemolysis takes place on standing. If this should 

 be the case to any marked extent, the antigen probably possesses 

 hemolytic properties in itself and is then likewise undesirable. 

 Formerly when simple alcoholic extracts were almost exclusively 

 in use this was a not infrequent occurrence, but with the extracts 

 prepared as described above, and in particular with the Noguchi 

 and the cholesterinized extracts, it is uncommon. 



2. The Hemolytic Amboceptor. To prepare the hemolytic ambo- 

 ceptor a large rabbit is injected on two occasions, seven days apart, 

 with the washed corpuscles corresponding to 30 c.c. of sheep's 

 blood, which must be obtained under aseptic precautions, and after 

 removal of the serum by centrifugation, washed with at least three 

 changes of sterile 0.9 per cent, salt solution. Care should be had 

 each time, after packing down the corpuscles by centrifugation 



