Studies of the alternate pathway in chelated serum

Activation of the alternate pathway in chelated serum by cobra venom factor (Co F), endotoxin of Escherichia coli, inulin, and zymosan was studied by immunoelectroprhoresis, radial immunodiffusion, and complement fixation tests. In serum chelated by 10 mM EGTA and 10 mM MgCl-2 (10 mM MgEGTA) activation by the classical pathway was blocked, but activation of C3PA and C3 occurred normally. By radial immunodiffusion assay, consumption of C3 and properdin was demonstrated to be similar to consumption in nonchelated sera. By immunoelectrophoresis, C3PA and C3 were shown to be activated by CoF in serum chelated with 6 mM but not with 10 mM EGTA without added Mg++. Inulin and endotoxin did not result in activation of either C3PA or C3, while C3PA but not C3 was activated by zymosan in the presence of 10 mM EGTA without Mg++ added. Consumption of properdin and C3, measured by radial immunodiffusion, was inhibited by 6 to 10 mM EGTA following activation by inulin, but not following activation by zymosan. The usefulness of 10 mM MgEGTA-chelated serum for the selective investigation of the alternate pathway in chemotaxis was demonstrated.