sodium-dodecyl-sulfate and Anemia--Hemolytic--Autoimmune

The factors contributing to RBC agglutination are complex. The RBC cytoskeleton's participation in and contribution to this phenomenon are difficult to separate from those of the plasma membrane. Immunoreactive, cytoskeleton-free, band 3-enriched microvesicles can be generated from normal RBCs. Band 3 has been defined as an important antigen in autoimmune hemolytic anemia (AIHA).. RBC microvesicles devoid of major cytoskeletal proteins were generated and sensitized with eluates obtained from AIHA patients, DAT-positive blood donors, and antisera to common RBC antigens. Monoclonal anti-human IgG was added and agglutination was investigated. Autoantibody-specific binding was evaluated by employing (125)I protein A.. RBC vesicle agglutination with a 4+ anti-human globulin score was obtained with 10 autoantibody eluates from AIHA patients and anti-D (3+), but not with eluates from 20 DAT-positive blood donors or antisera directed to eight other common RBC antigens. Microvesicles sensitized with AIHA eluates bound 67 to 167 times as much (125)I protein A radioactivity as did those incubated with buffered normal saline and 18 to 45 times more than vesicles incubated with normal serum.. The major proteins of the RBC cytoskeleton are not required for supporting IgG immune-mediated agglutination of RBC microvesicles.

Topics: Anemia, Hemolytic, Autoimmune; Anion Exchange Protein 1, Erythrocyte; Antibody Specificity; Autoantibodies; Coloring Agents; Cytoskeletal Proteins; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Hemagglutination; Humans; Indicators and Reagents; Rosaniline Dyes; Sodium Dodecyl Sulfate; Vacuoles

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.. 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.. 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.. 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.

Topics: ABO Blood-Group System; Anemia, Hemolytic, Autoimmune; Blood Proteins; Coombs Test; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Humans; Immunoglobulin G; Immunosorbent Techniques; Isoantibodies; Rh-Hr Blood-Group System; Sodium Dodecyl Sulfate