'58 



THE TECHNIQUE OF COMPLEMENT FIXATION. 



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

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

 into 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. Like in all biological reactions, 

 the quantitative relationship of these various ingredients determines to a great extent 

 the final result of the complement fixation test. As for antigen and antibody the experi- 

 ments of Weil and Nakayama must be considered; these are to the effect that only 

 one-half of the maximum non-hemolytic dose of each ingredient 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 laboratory 

 i c.c. of the dilution 1:10 represents the quantity chosen. Of the hemolysin the two 

 fold or three fold titre dose is taken and of the erythrocytes i c.c. of a 5 per cent, sus- 

 pension in normal saline solution suffices. Each of the five elements is diluted with saline 

 to make up i c.c. so that at the completion of the test all the tubes contain 5 c.c. Quite 

 a difference arises if an individual test is performed with a constant quantity of serum 

 and diminishing 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 + hemolysin + blood, to prove that the 

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



2. The double quantity of serum + complement + hemolysin + blood, to prove that the 

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



3. The "system control"; blood + 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 substituted 

 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. 



Titration of a Meningococcus Serum Obtained from the Horse, 

 a. Diminishing Quantities of Antigen. 



