294 1MMUNOLOGICAL METHODS OF DIAGNOSIS 



would represent its titer. After this has been ascertained 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 com- 

 plete at the expiration of thirty minutes in the water-bath. Occa- 

 sionally it happens that partial fixation of complement occurs with 

 normal serum even. Such antigens are evidently not fit for use, 

 for although every serum possesses anticomplementary properties 

 to a certain extent, it would be dangerous 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 it is uncommon. After the antigen has been tested in these 

 various directions it should be kept in the dark and preferably in 

 the ice-box. It will then not change its titer for a number of months, 

 but should not be looked upon as a stable product. In my labora- 

 tory we test the titer about once a month, and have reason to urge 

 this rule upon others. 



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 sheeps' 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 and pipetting 

 off the washings, to stir up the corpuscles in the new portion of saline 

 that is added. Finally, the corpuscles are suspended in such an 

 amount of saline that the volume injected equals that of the full 



