174 TECHNIQUE OF THE COMPLEMENT FIXATION METHOD 



c. Complement is obtained by killing a guinea-pig and using its serum 

 while fresh. The serum preserved in "Frigo" is, according to Sterns, not 

 reliable. 



d. Hemolysin is represented by the inactivated serum of a rabbit that 

 has been immunized against sheep's red blood cells. 



e. The twice washed sheep's red blood cells are used as erythrocytes. 



These five substances are placed in the test-tubes in the following order: antigen, 

 inactivated antiserum, complement; they are thoroughly mixed by shaking and 

 placed in the incubator for one hour in order to hasten their union. After this interval 

 the inactivated hemolysin and the red blood cells are added as indicator. The mixtures 

 are again returned to the incubator to promote hemolysis. As in all biological reac- 

 tions, the quantitative relationship of these various ingredients determines to a great 

 extent the final result of the complement fixation test. As far as antigen and anti- 

 body are concerned, the experiments of Weil and Nakayama must be considered; 

 these are to the effect that only one-half of the maximum dose of each ingredient 

 which does not bind complement is employed. With this point in view, preliminary 

 tests determining the proper dosage of each must be performed. 



The amount of complement used is always constant. In Wassermann's labora- 

 tory i c.cm. of the dilution i : 10 represents the quantity chosen. For most tests this 

 quantity is sufficient as it represents about three times the titer of normal guinea-pig's 

 serum. In certain instances it is preferable to work with smaller quantities, as in 

 Marmorek's method of complement fixation with the urine of tuberculous patients. 

 Of the hemolysin the two -fold or three-fold titer dose is taken and of the erythrocytes 

 i c.cm. of a 5 per cent, suspension in normal saline solution suffices. For Marmorek's 

 test the hemolysin is employed in just the titer dose, and of the red blood cells only 

 0.3 c.cm. is taken. Each of the five elements is diluted with saline to make up i c.cm. 

 so that at the completion of the test all the tubes contain 5 c.cm. Quite a difference 

 arises if an individual test is performed with a constant quantity of serum and dimin- 

 ishing doses of bacterial extract or reversely. Important tests should be carried out 

 by both methods. The necessary controls are: 



1. The double dose of antigen + complement -f hemolysin -f blood, to prove that 

 the dose of antigen employed in the test is correct (Weil and Nakayama). 



2. The double quantity of serum + complement + hemolysin -f- blood, to prove 

 that the dose of serum employed is correct (Weil and Nakayama). 



3. The "system control"; blood -f complement + one-half amount of hemolysin, 

 to show that the test was performed with double the hemolytic dose. 



4. Blood + salt solution, to prove that the salt solution is isotonic. 



In addition, it is advisable to repeat the test with inactivated normal serum sub- 

 stituted for the immune serum and another with a foreign instead of a homologous 

 antigen. 



These controls assure beyond doubt the specificity of the reaction. 



The accompanying chart represents schematically all that has been discussed. 



