EAC14 and EAC4 Preparation with a Magnesium-binding Chelator, and SAC4 Inactivation by a Factor in Normal Serum

Improvements have been made in the use of triethylenetetramine hexaacetic acid (TTHA), a strong chelator of Mg⁺⁺ but not of Ca⁺⁺, such that highly active EAC14 and EAC4 can be prepared by incubation of EA with TTHA-serum. It is essential to use a low ionic strenth, a long incubation period at 0°C, and to maintain a low pH (5.8) to avoid C3 contamination. The resulting cells are appreciably more active with limiting C2 than are cells prepared by the EAC1 plus EDTA-serum method, and stable to storage. IgG, IgM, and whole anti-Forssman antiserum all gave good preparations.

An unusual pH dependence was observed, in that SAC14 activity was generated between pH 5.6 and 7.0, and between pH 7.8 and 9.0, but not at pH 7.5. It was found that normal guinea pig serum contains a heat-stable factor which inactivates SAC4 at pH 7.5 in the presence of EDTA. This inactivation is such that there is a marked decrease in lysis with limiting C2, but no shortening of the T_max.