Concentration-Dependent Reactivity Profiles of a Human Monoclonal Antibody Recognising HLA-DQB1*04/05/06 and at Reduced Intensities to DQB1*03 Antigens.

Identifying HLA amino acid residues that account for an observed specificity (reactivity profile) is critical for serological categorisation at the B-cell epitope level. Human monoclonal antibody, LB_DQB0402_A, produced by an individual typed DQA1*03:03, DQA1*05:05/*05:11, DQB1*03:01, DQB1*03:01 against an immuniser typed DQA1*01:02, DQA1*05:05, DQB1*03:01, DQB1*06:02, reacts strongly to DQB1*04/05/06 single antigen beads (SABs), for which 55R, present in DQB1*04/05/06 alleles, has been previously proposed to be the target eplet. In this study, the antibody was also observed to react with DQB1*03 SABs albeit with reduced intensities and only at antibody concentrations higher than 1 μg/mL. Additional weak reactivities to DQB1*02 SABs were also observed when the concentration was further increased to 10 μg/mL. Different clusters responsible for the three concentration-dependent reactivity profiles were predicted by AminoAcid3D algorithm, and two (DQB1_46V and DQB1_55R) were verified using engineered variants (EVs) derived from DQA1*02:01-DQB1*06:01 expressed on cells and immobilised on beads, with bead assay more sensitive than cell assay. However, EVs targeting the third cluster (DQB1_81H) did not impact binding, suggesting 81H is not involved in binding to DQB1*04/05/06 when strong interacting 46V and 55R are present. On the other hand, 81H was predicted to account for the weak interactions with DQB1*02 antigens when neither 46V nor 55R are present. As to DQB1*03, only 46V and 81H are present, so the reduced signals could be due to the missing 55R. This antibody appears to pivot toward the stronger interacting residues available on different antigens. Although HLA alloantibodies develop and affinity mature against a specific foreign conformation, reactivity spread is expected if another antigen shares the same essential residue/region for specificity (ERS) and similar constituents of an epitope. However, this study demonstrates that reactivities in an in vitro setting can still occur even to antigens, including self, not carrying the same ERS as the immuniser, albeit of a much weaker intensity only observed above certain high antibody concentrations. While the clinical relevance of such observations remains unknown, this level of differentiation impacts the analysis of antigenicity.