272 INFECTION AND IMMUNITY 



quantity necessary to produce complete hemolysis of 1 c.c. of a five 

 per cent emulsion of blood cells, in the presence of two units of ambo- 

 ceptor. 



2. Hemolytic amboceptor: rabbit serum hemolytic for sheep cor- 

 puscles. Inactivated and titrated as for Wassermann test. 



3. A five-per-cent emulsion of sheep corpuscles in salt solution, pre- 

 pared as for Wassermann test. 



4. A highly potent typhoid antiserum obtained from an immunized 

 rabbit. In this case the smallest quantity of the immune serum which 

 will cause the fixation of complement in the presence of an emulsion or 

 extract of known typhoid bacilli is determined by experiment. 



It is best to rub up the centrifugalized bacteria with dry salt, adding 

 distilled water to isotonicity. When the extract is made, its anticom- 

 plementary dose is determined and the minimum quantity which in the 

 presence of known antityphoid serum will fix complement. These pre- 

 liminary titrations are analogous to those described as preliminary to 

 the W,assermann test. When these quantities have been determined, 

 an amount of the bacillary extract (about 1-3 or 1^4 of the anticom- 

 plementary dose) is chosen for the actual tests. It is well, also, in such 

 tests to determine the amount which will fix complement in the pres- 

 ence of a known normal serum, since occasional presence of antibodies 

 against some bacteria in normal serum may otherwise confuse the test. 

 The quantities of antigen and complement must then be chosen for the 

 test in such proportions that no fixation will occur with normal serum. 



5. Serum from the patient, inactivated at 56 C. for twenty 

 minutes. 



In the actual test a series of tubes are prepared each of which con- 

 tains: 



1. Complement, the determined quantity. 



2. Antiserum, the determined quantity. 



3. Diminishing quantities of the inactivated serum to be tested for 

 antigen beginning with 1 c.c. 



Salt solution is added for dilution to 3 c.c 



These substances are left together at 37 to 40 C. for one hour and 

 then the required quantities of amboceptor and red cells are added. 

 The reaction is controlled by tubes containing the same ingredients 

 without the typhoid antiserum. In a positive test there will be no 

 hemolysis in the tubes containing the patient's serum. 



