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Human monoclonal antibody against Rh(D) antigen: partial characterization
of the Rh(D) polypeptide from human erythrocytes
C Bloy, D Blanchard, P Lambin, D Goossens, P Rouger, C Salmon and JP Cartron
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.
Volume 69,
Issue 5,
pp. 1491-1497,
05/01/1987
Copyright © 1987 by The American Society of Hematology
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