470 COLLECTED STUDIES IN IMMUNITY 



chemical means into the analogous deflection of complement observed 

 with cobra amboceptor. 



I. Preparation of Cobra Lecithids. 



Owing to the tenacious character and the slight solubility of lecithin 

 in water it was, of course, impossible to attempt to effect the desired 

 combination by direct mixture of the aqueous solution of cobra 

 venom with lecithin. On the contrary it was necessary to adopt a 

 common chemical procedure, "shaking out," whereby, through the 

 agency of an appropriate solvent, the lecithin could combine with 

 the cobra venom. After a number of trials we found the best solvent 

 for this purpose to be chloroform. 



In our experiments we employed dried cobra venoms which had 

 kindly been placed at our disposal by Dr. Lamb and Dr. Greig of 

 Bombay, and Prof. Calmette of Lille. The lecithin used was the 

 so-called "Lecithol" of Riedel, and later on "Agfa-lecithin" of the 

 Actien-Gesellschaft fur Anilin-Fabrication. Both of these proved to 

 be excellent. Special emphasis must be laid on a sufficient purity 

 of the lecithin. For our purposes this is best recognized by testing 

 it against red blood-cells. 0.5 cc. of a 1% solution of the lecithin 

 should not dissolve red blood-cells. If the contrary is the case the 

 lecithin should be purified by precipitating it once or twice with 

 aceton. 1 



Forty cc. of a 1% solution of cobra venom in a 85%, salt solution 

 are mixed with 20 cc. of a 20%, solution of lecithin in chloroform. 

 The mixture is placed in a bottle holding about 100 cc. and thor- 

 oughly shaken for two hours in a shaking apparatus. Thereupon 

 the mixture is centrifuged for three-quarters of an hour in an electric 

 centrifuge making 3600 revolutions per minute. If the procedure 

 has been successful the chloroform layer must then be distinctly 

 separated from the watery portion, only a very slight compact, 

 cloudy, intermediate layer being present. If the lecithin is not 

 sufficiently pure this separation will not take place. The watery 

 portion is separated from the chloroform layer by carefully pipetting 

 off the former. The chloroform layer, usually measuring about 



1 We were also able to activate cobra amboceptor with a brom-lecithin 

 which Dr. Bergell kindly placed at our disposal. This preparation proved less 

 active than lecithin, but it evidently possesses the power to unite with cobra 

 amboceptor to form a lecithid. 



