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In solid organ transplantation, formation of de novo donor‐specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA‐specific monoclonal antibodies (mAbs) were generated from HLA‐reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA‐EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II‐specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA‐DR‐specific eplets, and conversion of five eplets (one HLA‐A, one HLA‐B, two HLA‐DR, and one HLA‐DP), from provisionally verified to truly antibody‐verified. Finally, one HLA‐DQ‐specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA‐specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting. [ABSTRACT FROM AUTHOR]

Eplet 44KM is currently listed in the HLA Epitope Registry but does not adhere to the eplet definition of an amino acid configuration within a 3.5 Å radius. Eplet 44KM has been previously redefined to the antibody‐verified reactivity pattern 44K/150V/158V, based on reactivity analysis of monoclonal antibody VDK1D12. Since the three residues are always simultaneously present on common HLA alleles, methods to define which residue is crucial for antibody‐induction and binding are limited. In this proof‐of‐concept study, we performed site‐directed mutagenesis to narrow down the antibody‐verified reactivity pattern 44K/150V/158V to a single amino acid and defined 44K as the eplet or functional epitope of mAb VDK1D12. [ABSTRACT FROM AUTHOR]