322 



INFECTION AND IMMUNITY 



use 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 titratioii, 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 found 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 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, are added. 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 Wassermann. 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. 324.) 



