452 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



alcohol require higher dilution, as 1 part of extract with 19 parts of salt 

 solution. 



3. Plain alcoholic extracts of human, beef and guinea-pig heart like- 

 wise require high dilution, as, for example, extract, 1 part and salt 

 solution, 19 parts. 



4. Alcoholic extract of human heart, prepared after the method of 

 Boas (quick method), is used in lower dilution, as 1 part of extract with 

 9 parts of salt solution. 



5. The solution of lecithin and cholesterin used by Browning, 

 Cruickshank, and Mackenzie is diluted with seven parts of salt solution. 



6. Aqueous extracts of syphilitic liver and extracts of pallida culture 

 are used undiluted, or may require dilution with four parts of salt solu- 

 tion. 



J Although these emulsions mil keep for a few days if placed in the re- 

 frigerator, it is advisable to make up only the amount required for imme- 

 diate use } as freshly prepared emulsions are better than older ones. 



Method of Titrating Antigens. Three values are to be determined: 



1. The anticomplementary dose, or that amount of antigen that in 

 itself is capable of fixing or inactivating the complement. 



2. The hemolytic dose, or that amount of antigen that in itself is 

 capable of lysing red blood-cells. This action is probably due to the 

 presence of certain lipoids and alcohol. 



3. The antigenic dose, or that amount of antigen that serves to 

 absorb or fix a certain and constant dose of complement with a definite 

 amount of syphilitic serum. 



1. Anticomplementary Titration. The determination of the anti- 

 complementary dose is probably the most important, for if an extract 

 were used in an amount that was anticomplementary or capable of 

 fixing complement in a non-specific manner, all the tests would show 

 false positive reactions, regardless of whether the serum was from a 

 normal or from a luetic person. 



After determining this anticomplementary dose, in conducting the 

 main test the antigen may be used in one-fourth the amount. 



All tests are conducted with chemically clean and preferably sterile 

 test-tubes (12 cm. by 13 mm.) and graduated pipets. Accuracy in 

 measurements is very essential in performing all complement-fixation 

 work. 



A preliminary titration of the hemolysin is made, with the complement 

 and corpuscle suspension to be used in titrating the antigen, in order to 

 determine its hemolytic dose, i. e., to adjust the hemolytic system. If, 



