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

Abstract

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-HC1 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 up to for two years could be identified. red cell membrane; human specific antigen; electrophoresis; membrane solubilization In forensic practice, species-identification of blood stains found at the scene was performed by immunoassay with anti-human hemoglobin. Recently, human specificity of each of plasma proteins was investigated by immunochemical methods (James 1965;Esteves and Binaghi 1972;Bauer 1974). On the other hand, little information about the antigens on or in red cell membrane has been presented. It was well known that the animals immunized with red cells of different species produced heteroagglutinins of various titers, indicating that species specific antigens were contained in red cell membrane.Recent advances in solubilizing red cell membrane revealed some constituents with antigenic activity of blood group or of enzymes (Madly and Dunn 1976). Bjerrum and Lundahl (1974) and Bjerrum et al. (1975) analyzed antigenicity of constituents of human red cells by two dimensional electrophoresis in which 19 membrane-specific immunoprecipitates were demonstrated.In the present study, we compare the solubilizing ability of various solutions

Immunochemical investigations of Rh0(D) activity detected in band 3 of red cell membrane.

“…IEP with 1.2% agar-agarose (1:1) was carried out using veronal buffer. Crossed IEP was performed as reported by Iwasa et al (1982). D activity of preparations at every step of this experiment was examined by agglutination inhibition test using diluted anti-D, papainized red cells and microtitrating plate (V type) .…”