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 RHDVI 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.