A human monoclonal anti-D antibody which detects a nonconformation-dependent epitope on the RhD protein by immunoblot.
We describe the first human monoclonal anti-D (LOR-15C9) which reacts with a D-specific motif exposed either on a native form on intact D-positive red cells or on a denatured form of the RhD protein (33 kD), and detected by immunoblotting. LOR-15C9 was able to precipitate RhD but not RhcE proteins produced by in vitro transcription-translation assays. The reactivity of the antibody, using panels of red cells with various partial D phenotypes known to lack some D epitopes and corresponding in RHD gene variants, suggested that LOR-15C9 reactivity depends on the portion of the RhD polypeptide encoded by the exon 7 (amino acids 314-358). These findings correlate well with the reactivity of LOR-15C9 with erythrocytes of some nonhuman primates (D(gor)-positive gorillas), but not of chimpanzee and Old or New World monkeys. In membrane proteins from partial D(VI) red cells, LOR-15C9 detected two proteins of molecular weight 33 and 21 kD: the presence of the latter was specific for category D(VI) and presumably represented the product of an alternatively spliced RHD(VI) transcript in these cells. This is consistent with the finding that LOR-15C9 can precipitate a shortened D protein mutant resulting from in vitro transcription-translation and lacking amino-acids 163-313 encoded by exons 4-6. In addition, a 21 kD band polypeptide was detected by immunoblot in all red cell samples but D--, using a rabbit anti-Rh polypeptide antibody (MPC8) raised against the C-terminal domain of Rh proteins. This 21 kD polypeptide most probably results from the translation of an alternatively spliced RHCE gene transcript. This study demonstrates that LOR-15C9 detects an epitope on the RhD protein that is independent of the membrane environment, and therefore could be a useful tool for the study of RhD polypeptides.
Apoil
P A
PA
Laboratoire d'Immunogénétique Moléculaire, Université Paul Sabatier, Pavillon Charles Lefebvre, Hôpital Purpan, Toulouse, France.
Reid
M E
ME
Halverson
G
G
Mouro
I
I
Colin
Y
Y
Roubinet
F
F
Cartron
J P
JP
Blancher
A
A
D000911:Antibodies, Monoclonal / Q000032:analysis; D015153:Blotting, Western; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007518:Isoantibodies / Q000032:analysis*; D010641:Phenotype; D010980:Platelet Membrane Glycoproteins / Q000276:immunology; D012204:Rh-Hr Blood-Group System / Q000276:immunology*; D018029:Rho(D) Immune Globulin
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007518:Isoantibodies; D010980:Platelet Membrane Glycoproteins; C061961:RHO(D) antibody; D012204:Rh-Hr Blood-Group System; D018029:Rho(D) Immune Globulin