HUMAN MONOCLONAL-ANTIBODY AGAINST RH(D) ANTIGEN - PARTIAL CHARACTERIZATION OF THE RH(D) POLYPEPTIDE FROM HUMAN-ERYTHROCYTES

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  • 1 May 1987
    • journal article
    • research article
    • Vol. 69  (5) , 1491-1497
Abstract
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 .times. 105, 0.43 .times. 105, and 0.32 .times. 105 antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of .apprx. 0.6 .times. 108 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 .times. 105/cell) and a high binding constant (5.7 .times. 108 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 .ltoreq. 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.