STANDARDIZING BACTERIAL ANTIGENS 475 



cent suspension of dried material in isotonic saline solution. . This 

 stock suspension is not filtered or centrifuged, but is further diluted 

 with saline solution, and constitutes the antigen (method of Besredka, 

 modified by Gay). The actual amounts of dry antigenic substance 

 contained in 1 c.c. of various dilutions are as follows: 



1 c.c. of 1: 40 dilution = 0.5 mg. 



1 c.c. of 1: 80 dilution = 0.25 mg. 



1 c.c. of 1: 160 dilution = 0.125 mg. 



1 c.c. of 1: 320 dilution = 0.062 mg. 



1 c.c. of 1: 640 dilution = 0.031 mg. 



1 c.c. of 1: 1280 dilution = 1.0155 mg., etc. 



Standardizing Bacterial Antigens. After an antigen has been pre- 

 pared it is standardized by determining the anticomplementary dose 

 i.e., the amount of antigen that just begins to show inhibition of hemol- 

 ysis due to non-specific complement fixation. This dose is easily 

 determined by adding increasing amounts of antigen to a series of test- 

 tubes with a constant dose of complement in each. As a general rule, 

 it is well to add to each tube a constant dose of fresh normal inactivated 

 serum, e. g., as 0.1 to 0.2 c.c., when the anticomplementary action of 

 serum alone is allowed for. I would emphasize the necessity of doing 

 this in experimental work with rabbit, dog, or any other animal serum. 

 After incubating for one hour, one and a half units of hemolytic ambo- 

 ceptor and 1 c.c. of corpuscle suspension are added to each tube, and the 

 tubes are reincubated for an hour or two and the reading made. In 

 the main test, one-quarter to one-half the anticomplementary unit may be 

 used, as this amount is known to be free from any power of non-specific 

 complement fixation. The former dose is, of course, safer than the latter. 



The standardization may be completed by determining the antigenic 

 dose of the antigen by titrating with a suitable and constant dose of 

 specific immune serum. This titration is conducted by placing in a 

 series of test-tubes increasing doses of antigen with a constant dose of 

 heated immune serum (usually 0.1 c.c.) and a constant dose of comple- 

 ment. After an hour the proper dose of hemolytic amboceptor and cor- 

 puscles is added. The readings may be made an hour or two later, or 

 after the tubes have been allowed to settle in a refrigerator. That tube 

 showing just complete inhibition of hemolysis contains the antigenic dose. 

 For the main test, it is well to use double this amount, providing this 

 dose is not more, and preferably less, than half the anticomplementary 

 dose. 



The antigenic titration is not always satisfactory, for when an arti- 

 ficial immune serum is used, the concentration of antibodies may yield 



