RUTH GILBERT 843 



tested for anticomplementary and hemolytic properties, as well as antigenic properties. If 

 found to be satisfactory, the antigen is diluted so that o.i cc. contains more than four anti- 

 genic units. 



The period of fixation is one hour at 37° C, and after the addition of the hemolytic 

 system, the tests are again incubated for two hours at the same temperature. 



Either i cc. of a i per cent suspension or o.i cc. of a 10 per cent suspension of human 

 blood cells is used in the hemolytic system with two units of antihuman rabbit serum as 

 amboceptor. 



The results are based on the percentage of cells that are hemolyzed, the tinting of the 

 salt solution, and comparison with complete inhibition and complete hemolysis. 



METHOD USED BY KOLMER, IN THE UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA^ 



The patient's serum is inactivated for fifteen minutes at 55° C. Graded amounts, 0.1, 

 0.02, 0.004, 0.002, and o.ooi cc, are used in a quantitative test. 



The complement consists of pooled guinea pig serum titrated with two units of anti- 

 sheep amboceptor. The amount of amboceptor as well as complement is adjusted each day. 

 Complement in varying amounts of a 1:30 dilution is titrated in the presence of antigen, 

 and the unit is determined after one hour in the water bath at 38° C. Slightly more than the 

 actual unit is used as the full unit to allow for the difference in the method of fixation. Two 

 units are used in the test. 



The antigen is a lecithinized alcoholic extract of beef- or human-heart muscle from which 

 the acetone-soluble substances have been removed and 0.2 per cent cholesterin added. 

 Dilutions are made by slowly adding the extract to salt solution. Titrations for herriolytic, 

 antigenic, and anticomplementary properties are made. Ten antigenic units are used, and 

 twenty times this amount should not be anticomplementary or hemolytic. The antigen is 

 diluted so that the proper amount for the test wiU be contained in 0.5 cc. The serum and 

 antigen are allowed to stand from five to thirty minutes before the addition of complement. 



From fifteen to eighteen hours at from 6° to 8° C. are allowed for fixation, followed by 

 from five to fifteen minutes in the water bath at 38° C. Amboceptor and cells are added 

 separately. The period for secondary incubation is one hour at 38° C. The tests are then 

 placed in the refrigerator to allow the cells to settle, and the results are recorded from one to 

 three hours later. 



In determining the reactions, a scale containing antigen, inactivated complement, and 

 varying amounts of hemoglobin solution, and cell suspension is used for comparison. 



METHOD USED IN THE NEW YORK STATE DEPARTMENT OF HEALTH,^ 

 DIVISION OF LABORATORIES AND RESEARCH 



The total volume of the test is 0.5 cc. The patient's serum, after inactivation for one- 

 half hour at 55° C, is tested in amounts of 0.05 and 0.02 cc. 



The complement is a mixture of the fresh serum from six or more guinea pigs. Each of 

 the sera has been found satisfactory by preliminary tests made before the pool is prepared. 

 The mixture is then titrated and diluted so that the two units used in the test are contained 

 in O.I cc. 



Two antigens are used: one, an acetone-insoluble antigen prepared and diluted accord- 

 ing to the method of Bordet and Ruelens, and an alcoholic extract reinforced with 0.4 per 



' Kolmer, J. A.: Infection, Immunity and Biologic Therapy (3d ed.). Philadelphia and London: 

 W. B. Saunders Co., 1923. 



2 Wadsworth, A. B. : Standard Methods of the Division of Laboratories and Research of the New York 

 State Department of Health. Baltimore: Williams & Wilkins Co., 1927. 



