Inhibition studies with recombinant anti-Rh(D) antibodies. Panels show results of representative experiments demonstrating the mutual inhibition of antibodies directed at 2 different Rh(D) epitopes (in this example, epD3 and epD6/7; A and C), but not between an Rh(D) antibody and an unrelated recombinant anti-RBC antibody (an anti-blood group B antibody; B). In (A), Rh(D)-positive RBCs were incubated with soluble Fabs only, phage-displayed Fabs only, or combinations of the two, as indicated. In (B), Rh(D)-positive RBCs that were blood group B were used. After washing, RBCs were resuspended in anti-M13 antibody and assessed for agglutination induced by phage-displayed Fabs. Soluble Fabs were used full-strength, whereas Fab/phage preparations were present in limiting amounts to increase the sensitivity of the inhibition assay (see the Materials and Methods). In (C), mutual inhibition of epD3 and epD6/7 anti-Rh(D) antibodies was demonstrated with Rh(D)-positive RBCs, γ1κ and γ1λ soluble Fabs, and light chain isotype-specific antisera (see text for details). In these examples, the anti-epD3 and anti-epD6/7 antibodies were clones E1/M3 and D5/I3, respectively. The anti-blood group B antibody was isolated from an IgG phage display library made from the splenic B cells of a blood group O donor.61
