CHAPTER XIV. 

 THE TECHNIQUE OF COMPLEMENT FIXATION. 



Original method of Bordet-Gengou. Wassermann-Bruck's modification. Technique 

 of serum diagnosis for syphilis. Echinococcus disease. Differentiation of proteids ac- 

 cording to Neisser-Sachs. 



I. The Original Method of Bordet-Gengou. 



a. The antigen consists of bacteria grown upon agar for twenty-four 

 hours and then suspended in physiological salt solution to make a rather 

 concentrated emulsion. 



For typhoid bacteria Bordet and Gengou take 5 c.c. of salt solution to each culture of 

 bacteria. 



For tubercle bacilli 80 mg. of the bacteria are suspended in i c.c. of salt solution. 



b. The serum containing the antibody is heated for one-half hour at 

 56 C. to destroy the complement. 



c. As complement, the fresh serum of a normal animal or human being 

 is used. 



d. The hemolysin is produced by the inactivated serum of a rabbit 

 that had been immunized against sheep's or goat's erythrocytes, or the 

 serum of a guinea pig injected with rabbit's red blood cells. 



e. The respective red blood corpuscles are washed, to free them of 

 their complement containing serum. 



A definite amount of bacterial suspension is mixed with varying amounts of inactivated 

 immune serum and a proportional amount of complement is added. These three ingre- 

 dients are mixed and allowed to remain at room temperature for four to five hours. 

 During this time the complement is fixed if the antigen and antibody are of a homologous 

 nature. In order to see whether this union has taken place or not, hemolysin and 

 erythrocytes are added in a mixture thus prepared: 2 c.c. of inactivated hemolysin + 

 twenty drops of washed blood cells are mixed and allowed to remain together for about 

 fifteen minutes so that the erythrocytes are sensitized, i.e., united with the hemolytic 

 amboceptor. Of this mixture each tube receives o.i to 0.2 c.c. If the complement 

 has not become fixed, hemolysis occurs in several minutes. If the complement has 

 become so, hemolysis does not occur; since, however, the hemolysin also contains 

 hemagglutinin, the erythrocytes are agglutinated and sink to the bottom of the tubes. 

 As control tests, Bordet and Gengou considered the following very necessary: 

 i. Bacterial suspension + inactivated normal serum (instead of immune serum) 

 + complement (five hours) + hemolysin + blood. Hemolysis must occur, as the 

 normal serum does not contain enough amboceptors to unite with the bacterial suspen- 

 sion and consequently complement remains unbound. 



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