title: Comparison of serum anti-band 3 and anti-Gal antibody binding to density-separated human red blood cells.
abstract: This study examines the quantitative relationship between two natural serum antibodies, anti-band 3 and anti-alpha-galactosyl (anti-Gal), in their capacity to bind to human red blood cell (RBC) populations separated on density gradients. The question was approached in two ways. First, we determined the extent of rebinding of affinity-purified human serum antibodies to RBCs that had been stripped of in situ antibody. Second, we eluted the in situ bound antibody at low pH from density-separated RBCs and determined the proportion of total eluted antibody that bound specifically to erythrocyte band 3 or to a Gal-alpha-(1,3)-Gal structure. Our results show that high-density human RBCs bind increased amounts of both antibodies. Anti-Gal rebinding was specific, because it was saturable and occurred in the presence of serum IgG depleted of anti-Gal. Binding assays using control natural autoantibodies directed against antigens not found on the RBC surface showed that high-density RBCs also bind increased amounts of these antibodies as compared with low-density RBCs. However, the extent of this binding is substantially lower than that of anti-band 3 and anti-Gal. Binding studies using the lectins Bandeiraea Simplicifolia (alpha-galactosyl specific) and Arachis Hypogaea (peanut agglutinin, beta-galactosyl specific) indicated that only the alpha-galactosyl sites are exposed on high density RBCs, and not the beta-galactosyl structure characteristic of T antigen. Antibody that is eluted at low pH from high density RBCs contains a 5.0% to 18.0% component that binds to band 3 protein, and a 9.1% to 39.0% component that recognizes the alpha-galactosyl structure. Together, the two antibodies appear to constitute an average of 35% (range 17.2% to 57.4%) of the in situ bound antibody from high-density human RBCs.
authors.affiliation: Department of Laboratory Medicine, University of California, San Francisco 94143.
authors.forename: M P
authors.initials: MP
authors.lastname: Sorette
authors.forename: U
authors.initials: U
authors.lastname: Galili
authors.forename: M R
authors.initials: MR
authors.lastname: Clark
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000276:immunology* / Q000378:metabolism; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism; D000937:Antigen-Antibody Reactions / Q000276:immunology; D002469:Cell Separation / Q000379:methods; D004912:Erythrocytes / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D037102:Lectins; D014312:Trisaccharides / Q000276:immunology* / Q000378:metabolism
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D000906:Antibodies; D007074:Immunoglobulin G; D037102:Lectins; D014312:Trisaccharides; C061732:3'-galactosyllactose