Epitope mapping of four monoclonal antibodies specific for the human RhD antigen

RhD is a highly immunogenic erythrocyte membrane protein, implicated in hemolytic disease of the newborn and other hemolytic disorders. Anti-RhD antibodies are used in the treatment of such disease states. Six mutant forms of recombinant RhD were stably expressed in K562 cells, and these cells were used to investigate epitope specificities of four anti-RhD monoclonal antibodies (mAbs). Amino acid substitutions were made in the exofacial loops of RhD to the corresponding residues found in the related RhCE polypeptide; M169L/M170R and I172F in the third loop, F223V and E233Q in the fourth loop, and D350H and G353W/A354N in the sixth loop. Each mAb was found to have a unique fine specificity and recognized multiple distant sites within RhD. The mAbs also differed in how they recognized individual amino acids in the exofacial loops of RhD.