An Improved Method for the Preparation of EAC14, an Intermediate in Immune Hemolysis by Carp Complement

We have succeeded in preparing EAC14 (sheep erythrocytes sensitized with carp antibody and complement components, Cl and C4) by incubating EA with carp serum in the presence of 5 mM TTHA (triethylenetetramine-N,N,N’,N’:N”:N”’hexaacetic acid), a selective chelator of Mg’+, and 5 mM CaCl* at pH 7.4. The optimal conditions for preparing EAC14 were as follows: ionic strength, 0.085; reaction temperature, 25°C; incubation period, 10 min. The EAC14 prepared by this method was stable for at least 4 days when stored at 4°C in GGVB*+ (veronalmbuffered saline, pH 7.4, containing 0.1% gelatin, 0.15 mM CaCl,, 0.5 mM MgCl,, 2.5% glucose and 0.02% NaN,).