An improved method for the isolation or erythrocyte antigen band-3

RESUMO

An improved method for isolation of human and Rhesus monkey band-3 separated by sodium dodecyl sulfate poliacrylamide gel electrophoresis (SDS-PAGE) is described. Purified band-3 was obtained from human hemoglobin-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 per cent NaHCO3, containing 1 per cent SDS, for 2h, with shaking, at room temperature, followed by overnight incubation at 4ºC. The preparation was subsequently sonicated and clarified by centrifugation. Supernatants were dialyzed against distilled water, their protein contents were measured, and the presence of purified band-3 was demonstrated by SDS-PAGE. A calibration curve was developed for assay of CE+S material using densitometric evaluation of the protein profile on SDS-PAGE. An amount of 37.5 mg of Hb-free ghost grave 3.15 mg of purified band-3 after CE+S, corresponding to an 8.4 per cent yield. Rabbits were immunized with 50µg CE+S antigen. Serawere collected and assayed by Western blot analysis against its proteolytic fragments, which were obtained from packed red blood cells by treatment with protease type VI from Streptomyces griseus (1h at 37ºC), followed by extensive washing and hypotonic lysis. Specific antibodies recognized band-3 and its proteolytic fragments 60 and 63 kDa in human ghosts obtained from different blood donors, confirming the genetic polymorphism. Analogous serum obtained against the Rhesus monkey band-3 proteolytic fragment 63 kDa recognized the human antigen and its respective fragments. These results indicate the existence 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 of band-3 and its fragments with satisfactory yield and maintenance of both their immunogenic and antigenic properties