AN IMPROVED METHOD FOR THE ISOLATION OF ERYTHROCYTE ANTIGEN BAND-3

An improved method for isolation of human and Rhesus monkey band-3 sep arated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (S DS-PAGE) is described. Purified band-3 was obtained from human hemoglo bin-free ghosts (Hb-free ghosts) after SDS-PAGE by chemical elution sonication (CE+S). The section of the gel corresponding to the antigen was cut out, mechanically disrupted and incubated in 1% NaHCO3 contai ning 1% SDS, for 2 h, with shaking, at room temperature, followed by o vernight incubation at 4 degrees C. The preparation was subsequently s onicated and clarified by centrifugation. Supernatants were dialyzed a gainst distilled water, their protein contents were measured, and the presence of purified band-3 was demonstrated by SDS-PAGE. A calibratio n curve was developed for assay of CE+S material using densitometric e valuation of the protein profile on SDS-PAGE. An amount of 37.5 mg of Hb-free ghosts gave 3.15 mg of purified band-3 after CE+S, correspondi ng to an 8.4% yield. Rabbits were immunized with 50 mu g. CE+S antigen . Sera were collected and assayed by Western blot analysis against its proteolytic fragments, which were obtained from packed red blood cell s by treatment with protease type VI from Streptomyces griseus (1 h at 37 degrees C), followed by extensive washing and hypotonic lysis. Spe cific antibodies recognized band-3 and its proteolytic fragments 60 an d 63 kDa in human ghosts obtained from different blood donors, confirm ing the genetic polymorphism. Analogous serum obtained against the Rhe sus monkey band-3 proteolytic fragment 63 kDa recognized the human ant igen and its respective fragments. These results indicate the existenc e of similarities between these two species of band-3, suggesting the potential use of this technique in taxonomic and phylogenetic studies. Purification by CE+S is an efficient and rapid method for isolation o f band-3 and its fragments with satisfactory yields and maintenance of both their immunogenic and antigenic properties.