(A) ELISA results showed the specificity of HD5 and HD6 to B272 homodimers when challenged for different recombinant HLA class I complexes (-A1,-B7,-B13,-C7,-B27, and-B272). Control HC10 and W6/32 antibodies were used as positive and negative control, respectively. (B) Direct ELISA against HLA-G and B272 homodimers using HD5, HD6, HC10 and W6/32 antibodies showed specificity of HD5 and HD6 to immobilized B272 homodimers but not to HLA-G homodimers. (C-E) Recombinant B272, B27-free-heavy chain and HLA-B27 heterotrimers were immobilized into chips for kinetic characterization by SPR. C) HD5 and ligand (B272) have a Kd of 0.32 nM. Immobilized free-heavy chains (D) or HLA–B27 heterotrimers (E) did not interact with HD5 and Kd values were not fitted. (F) Blocking competition experiments in SPR were performed by immobilizing B272 to a streptavidin chip followed by injection of HD5 to form B272-HD5 complexes. Injections of LILRB2, KIR3DL2 and Pirb were assessed and binding events recorded.