500 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



practical value. More recently von Dungern 1 has advocated a method 

 that yielded 90 per cent, of positive reactions in known cases of cancer. 

 Positive reactions have also occurred in tuberculosis and syphilis, and 

 the reports of various other investigators are somewhat contradictory. 

 Whitman, in a study of 30 cases, found the method highly satisfactory. 



Preparation of Antigen. Two different antigens may be employed. 

 Of these, von Dungen prefers the first. 



First Method. Place 10 c.c. of blood (preferably obtained from a 

 patient suffering from general paralysis) in a centrifuge tube containing 

 0.1 c.c. of a 20 per cent, solution of sodium oxalate. Wash the cells 

 three times with normal saline solution. After the last washing, measure 

 the corpuscles and add 20 parts by volume of chemically pure acetone. 

 Let this stand for three days at room temperature, giving it an occasional 

 shaking. Filter and evaporate the filtrate to dryness in the incubator, 

 and take up the residue at once with enough 96 per cent, alcohol to form 

 a 1 per cent, solution. This usually requires about 10 times as much 

 alcohol as the amount of acetone used. Before using, the alcoholic 

 solution is mixed slowly with four parts of normal saline solution and 

 titrated for its anticomplementary dose. Half this amount is used in 

 making the main test. 



Second Method. The tumor tissue, freed as much as possible of fat 

 and degenerated portions, is minced and treated with 20 volumes of 

 acetone and extraction continued for one or two weeks at room tempera- 

 ture with an occasional shaking. The extract is then filtered, evaporated 

 to dryness at 37 C., and the residue taken up with half the amount of 

 absolute alcohol. This extract is then diluted 1 : 10 or 1 : 20 with salt 

 solution, and titrated for its anticomplementary dose, one-half this 

 amount being used in making the main test. 



The Test. The serum should be fresh. Add two volumes of ^ 

 caustic soda (JQ soda diluted with four volumes of normal saline solu- 

 tion), and inactivate the mixture for one-half hour at 54 C. (not 56 C.). 

 The soda solution must be prepared accurately from chemically pure 

 substances, must be free from carbonate, and must be protected from 

 the air. 



Into a series of five test-tubes place 0.075, 0.15, 0.3, 0.6, and 0.6 c.c. 

 of the serum-soda mixture. To the first four tubes add the proper 

 amount of antigen. To all tubes add 1 c.c. of complement diluted 1 : 20 

 and sufficient salt solution to bring the total volume up to 3 or 4 c.c. 



1 Munch, med. Wochenschr., 1912, 59, 65, 1098, 2854. 



