Identification of Amino Acid Residues, Distal to the Peptide-Binding Domain, That Confer Serological Distinctions Within Each HLA-DQB1*02, *03, *05 and *06 Antigen Group.

Identifying HLA amino acid residues that account for an observed specificity (reactivity profile) is critical for serological categorisation at B-cell epitope level. Human monoclonal antibody, LB_DQB0501_C (hIgG1l), produced by an individual typed DQA1*01:02, DQA1*03:01, DQB1*03:02, DQB1*06:09 against an immuniser typed DQA1*01:01, DQA1*01:02, DQB1*05:01, DQB1*06:09, reacts strongly to many but not all DQB1*02, *03, *05 and *06 single antigen beads. Within each first-field molecular typing group, some beads were recognised weakly by the antibody or not at all even at 10 μg/mL. Only one prediction (DQB1_130R185T as a pair) was made using AminoAcid3D (AA3D), an algorithm that defines each HLA molecule as a series of overlapping 3-dimensional clusters and deduces the potential essential region that could account for an observed reactivity profile. Among various scenarios, DQB1_130R185T was predicted only when the weak signals (TMFI about 1000) were included as positive. Both 130R and 185T, about 10 Å apart, were confirmed as essential for binding using engineered variants as the change of either led to complete loss of binding in both cell and bead assays. Additional residues in the vicinity were also tested for their relative contributions. Besides polymorphic residues in DQB1 at positions 135 or likely 182 that could explain the signal differences within each DQB1*02 or DQB1*05 group, three conserved residues (DQB1_132F, _174H and _183P) were also shown to be critical in securing the binding, indicating multiple residues including conserved are needed to maintain a stable interaction. Epitopes distal to the peptide-binding domains have not been sufficiently described by the conventional serological typing. Even using HLAMatchmaker, 130R185T was not predicted despite both 130R (verified, level A2) and 185T (unverified) are listed separately as an HLA-DQ eplet in HLA Eplet Registry. This study provides evidence that AA3D is a useful research tool for epitope analysis. Once a predicted specificity determinant is experimentally verified, both polymorphic and conserved positions nearby should be further investigated for their contributions to the epitope. Such knowledge could become the basis of the ongoing efforts in serological categorisation.