Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes.
A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29-kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS-PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001402:B-Lymphocytes / Q000378:metabolism; D001666:Binding Sites, Antibody; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D003902:Detergents / Q000494:pharmacology; D004912:Erythrocytes / Q000032:analysis* / Q000648:ultrastructure; D004854:Herpesvirus 4, Human / Q000276:immunology; D006801:Humans; D011233:Precipitin Tests; D012204:Rh-Hr Blood-Group System / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification*; D012795:Sialoglycoproteins / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D003902:Detergents; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; D012795:Sialoglycoproteins