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The protein composition of red cell eluates

BACKGROUND: The protein composition of red cell (RBC) eluates has not been extensively studied. The purpose of this study was to determine the IgG content and protein composition of RBC eluates prepared by the acid and xylene elution methods. STUDY DESIGN AND METHODS: Six samples of group O R1R1 RBCs sensitized with anti‐D in vitro, six nonsensitized samples of the same group O R1R1 RBCs, and six samples from patients with warm autoimmune hemolytic anemia (WAIHA) were studied. The eluate protein composition was determined by sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis, and immunoglobulin concentrations were estimated by an immunoblot technique using horseradish peroxidase‐ conjugated anti‐IgG and 3,3′ diaminobenzidine. RESULTS: The protein concentrations of the xylene eluates were significantly greater than those of the acid eluates (37.3 +/− 10.7 and 3.0 +/− 0.4 [SD], respectively; p < 0.005). In all samples, the proportion of IgG was less than 0.13 percent of the total protein content. The acid eluates of sensitized RBCs contained more IgG than the xylene eluates. The antibody titers of eluates from WAIHA RBCs were significantly lower than eluates of in vitro sensitized RBCs (p < 0.005). The estimated molecular weights of the Coomassie blue‐stainable protein bands from xylene eluates were 97, 78, 63, 45, 31, 23, and 16 kDa, and those of bands from acid eluates were 97, 78, and 55 kDa. No periodic acid‐ Schiff reagent‐stainable bands were detected. CONCLUSION: These data indicate that IgG represented only a small fraction of the proteins in the eluates and that the protein composition varies with the elution procedure.