Immunoelectrophoretic analyses of antigenic and human specific proteins of red cell membrane

Rabbit antisera were raised for red cell stroma extracted with each of the following solutions; 5 mM phosphate buffer pH 8.0 (A antigen). 5 mM EDTA containing 5 mM 2-mercaptoethanol (B), 1/15 M phosphate buffered saline (C), 0.05% SDS (D), 50 mM Tris-HCl buffer pH 8.0 containing 1% SDS, 1 mM EDTA and 1% 2-mercaptoethanol (E) and 1% Triton X-100 (F). Crossed immunoelectrophoresis showed that E antigen produced relatively numerous precipitation lines, 7-9, against each of the antisera, suggesting that E solution was superior to the others for membrane solubilization. However, the Ouchterlony test demonstrated potent precipitin activity in anti-C, and the antiserum absorbed with monkey stroma which was extracted with F solution was proved to have strict human specificity. By adopting the antiserum to affinity chromatography, human specific antigens were isolated. The antigens gave four bands moving faster than Band 5 at SDS-polyacrylamide gel electrophoresis none of which was stained by Schiff's reagent. Using absorbed anti-C, human specificity of blood stains kept us to for two years could be identified.