Studies in Complement Fixation. 



in the case of the syphilitic sera, non-specific; four different 

 Wassermann antigens being employed with each serum. Three 

 fixation temperatures — water-bath, room and ice-box — were re- 

 sorted to. Some phases of this investigation are still in progress 

 and in this preliminary report, the work with the purified proteins 

 only will be reported, although our findings indicate that the rate 

 of fixation of complement is the same, no matter what type of 

 fixing antibody is used. 



Two purified proteins were employed: Edestin obtained from 

 hempseed and phaseolin obtained from the kidney bean. These 

 were kindly furnished by Dr. Thomas B. Osborne. Two rabbits 

 were immunized with edestin and two with phaseolin. In order 

 to elicit quantitative differences in the antibody production in the 

 rabbits, four modes of immunization were resorted to. The 

 edestin rabbits were injected intravenously according to "Im- 

 munization Methods No. i and No. 2," respectively, described 

 by Kahn and McNeil in another paper. 1 The phaseolin rabbits 

 were injected intraperitoneally. One rabbit received 100, 150, 

 200, 250 and 300 mgm. of phaseolin at 48-hour intervals, and the 

 other 100, 150 and 200 mgm. of this protein at 24-hour intervals. 



The complement fixation experiments were carried out in 

 one tenth quantities of regular Wassermanns, otherwise in the 

 usual manner, with 2 units of complement, 2 units of amboceptor 

 and 0.1 c.c. of a standard 5 per cent, suspension of sheep-cells. 

 The respective antigens were prepared by weighing out 10 mgm. of 

 the protein and dissolving these in 10 c.c. of iV/1000 NaOH to 

 which was added 0.05 c.c. of N/10 NaOH. The alkali was neces- 

 sary in order to get the proteins in solution. One c.c. of this 

 protein solution was added to 9 c.c. of saline and 0.1 c.c. of this 

 final solution (0.01 mgm. of the protein) was used in the tests. 

 The serum dilutions employed in the tests were the following: 

 0.01 c.c, 0.007 c.c, 0.004 c.c, 0.003 c.c, 0.002 c.c, 0.001 c.c, 

 0.0005 c.c, 0.0003 c.c, and 0.0001 c.c 



After establishing the presence of specific complement fixing 

 antibodies in the rabbit's sera by preliminary tests, fixation experi- 

 ments were carried out with the serum dilutions indicated above, 

 varying both the lengths of time and the temperatures of fixation. 



1 /. Immunol, 1918, iii, 281. 



