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

Abstract

Stroma from D positive red cell was solubilized with 1% deoxycholic acid (DOC) and was applied onto Sepharose 4B and 6B, followed by fractionation with activated thiol Sepharose 4B. By this method Band 3 containing no carbohydrate was prepared. The Band 3 was incubated with human anti-D, then immune complex soluble in DOG solution was produced. This complex was not prepared from red cell components other than Band 3, nor from Band 3 from D negative red cell. The complex agglutinated papainized D positive red cells. Molecular weight of the complex was estimated to be 500,000 to 700,000 by the gel filtration method. The molecular weight increased according to the time of incubation. The complex gave a precipitation line in counter immunoelectrophoresis (IEP), a single peak in crossed IEP against anti-Band 3, and a line at y-globulin region in IEP against anti-human serum. SDSpolyacrylamide gel disc electrophoresis of the complex showed the pattern consisting of fragments of immunoglobulin G and Band 3 which was not stained with periodic acid Schiff reagent. These support our results that Rh-Hr blood type activities are located in Band 3 almost free of carbohydrate.Rh-Hr antigens; Band 3; immune complexOf major blood types, such as ABH, Rh-Hr, MN, P, I and Lewis, only Rh-Hr has many problems to be solved. These Rh-Hr antigens are known to locate on the membrane of human red cells but not on membrane of other tissues of human body (Mollison 1979). Characterization of Rh-Hr antigens has been frustrated by the inability to solubilize red cell membrane without loss of the antigenicity. SDSpolyacrylamide gel disc electrophoresis (SDS-PGDE) revealed that red cell membrane is composed of about 20 components with different molecules (Abraham and Bakerman 1975). In a previous paper (Yokoi et al. 1983), we described the isolation and purification of Rho(D) antigen, which located in Band 3 with no periodic acid Schiff (PAS) reaction. In this paper, the immune complex of anti-D and D active Band 3, which was prepared from deoxycholic acid (DOC) solubilized stroma, was investigated.Reagents and antisera. Sephadex G-25, Sepharose MATERIALS AND METHODSResins for gel filtrations and ion-exchange chromatographies, 4B, 6B and activated thiol Sepharose 4B were obtained

Preparation of anti-Lea and anti-Rho (D) sera by immunization with blood group substance trapped in autologous red cell ghost.

“…Band 3 (protein concentration 4.0 mg/ml) was mixed with a half volume of human anti-D (Ortho Diagnostics, New Jersey, USA) and the resulting immune complex was isolated by gel filtrations (Yokoi et al 1983c). Five animal groups (Groups 3, 4, 5, 6 and 7) were used for preparaing anti-D.…”