Haemolysis of human erythrocytes heavily sensitized with sheep amboceptor by sheep complement chelated with EGTA or Mg2+-EGTA

Human erythrocytes sensitized with an agglutinating dose of either sheep anti-human erythrocyte antiserum, IgM, slow or fast γ-globulin antibody are haemolysed by foetal lamb or sheep serum complement (C) in the presence of ethyleneglycoltetraacetic acid (EGTA) or Mg²⁺-EGTA but not ethylenediaminetetraacetic acid (EDTA) or triethylenetetraminehexaacetic acid (TTHA). Dialysis of the C source against phosphate buffered saline rendered it inactive, but addition of Mg²⁺ or Mg²⁺-EGTA restored haemolytic activity. Haemolysis of sensitized erythrocytes took place more rapidly in buffer containing Ca²⁺ and Mg²⁺ than in EGTA or Mg²⁺-EGTA. Heating the C source at 50 °C for 30 min or 56 °C for 3 min abolished haemolytic activity. In the presence of EGTA or Mg²⁺-EGTA, haemolytic activity was restored to 50 °C-heated serum by a CM-cellulose fraction that contained sheep factor B.These results show that sheep C, in the presence of EGTA or Mg²⁺-EGTA will haemolyse human erythrocytes heavily sensitized with sheep amboceptor. The alternative pathway of sheep C is thought to be involved.