268 INFECTION AND IMMUNITY 



for the test, the patient's serum must be inactivated by heating in a 

 water bath to 56 C. for twenty minutes to half an hour. 



THE TEST. The actual test for antibody in a suspected serum is 

 carried out in the following way: In a test-tube of suitable size, 2 units 

 of the complement, 0.2 c.c. of the inactivated suspected serum, and the 

 antigen, in quantity determined by titration, are mixed, and the total 

 volume brought up to 3 c.c. with normal salt solution. This mixture is 

 thoroughly shaken, and placed for one hour in a water bath or in the 

 incubator at 37.5 C. Recently it has been fourid that more delicate 

 results are obtained when the fixation is allowed to take place in the 

 refrigerator for three or four hours the so-called "ice-box method." 

 At the end of this preliminary incubation there is added 1 c.c. of a 5 

 per cent emulsion of sheep's corpuscles, and two units of hemolytic 

 amboceptor, determined by a titration of the inactivated hemolytic 

 rabbit serum, as described above. This mixture is again placed at 37.5 

 C. for one to two hours. If the antibody is present in the suspected 

 serum, no hemolysis takes place. If absent, hemolysis is complete. 



In our own work all tests are done in half the quantities of the 

 original Was'sermann. Hence only 0.1 c.c. of the patient's serum, and 

 the antigen and complement as determined in titrations with 0.5 c.c. 

 of the cells are mixed in a total volume of 1.5 c.c. At the end of the 

 preliminary incubation, 0.5 c.c. of cells previously sensitized with 2 

 units of amboceptor are added. 



No test is of use unless suitable controls are made. The controls 

 set up should be as follows: 



Control 1. For each serum tested the mixture described above, 

 omitting antigen. 



Controls 2 and 3. The mixture made as in the test but with known 

 syphilitic serum (2) with and (3) without antigen. 



Controls 4 and 5. The mixture made as in the test, but with normal 

 serum (4) with and (5) without antigen. 



Controls 6 and 7. The hemolytic system, complement, blood cells 

 and amboceptor, set up in order to show that the system is in working 

 order (6) with and (7) without antigen. It is convenient to set the 

 tubes in two rows in a rack, the front row containing antigen, the back 

 row containing the same mixture without antigen. 



In a positive test, the test itself, and Control 2, alone, should show 

 inhibited hemolysis. The other tubes should show complete solution 

 of the hemoglobin. (See scheme, p. 259.) 



Modifications of the Wassermann Test. Since the original for- 



