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Matching strategies based on HLA eplets instead of HLA antigens in solid organ transplantation may not only increase the donor pool for highly sensitized patients, but also decrease the incidence of de novo donor-specific antibody formation. However, since not all eplets are equally capable of inducing an immune response, antibody verification is needed to confirm their ability to be bound by antibodies, such that only clinically relevant eplets are considered. The HLA Epitope Registry has documented all theoretically defined HLA eplets along with their antibody verification status and has been the foundation for many clinical studies investigating eplet mismatch in transplantation. The verification methods for eplets in the Registry range from polyclonal sera from multi- and uni-parous women to murine and human monoclonal antibodies (mAbs), and antibodies purified by adsorption and elution from sera of HLA immunized individuals. The classification of antibody verification based on different methods for validation is problematic, since not all approaches represent the same level of evidence. In this study, we introduce a classification system to evaluate the level of evidence for the antibody-verified status of all eplets in the HLA Epitope Registry. We demonstrate that for a considerable number of eplets, the antibody-verified status is solely based on polyclonal serum reactivity of multiparous women or on reactivity of murine mAbs. Furthermore, we noted that a substantial proportion of patient sera analyses and human mAb data presented in the HLA Epitope Registry Database has never been published in a peer-reviewed journal. Therefore, we tested several unpublished human HLA-specific mAbs by luminex single antigen beads assay to analyze their HLA reactivity for eplet antibody verification. Although the majority of analyzed mAbs indeed verified their assigned eplets, this was not the case for a number of eplets. This comprehensive overview of evidence for antibody verification of eplets in the HLA Epitope Registry is instrumental for future investigations towards eplet immunogenicity and clinical studies considering antibody-verified eplet mismatch in transplantation and warrants further standardization of antibody verification using high quality data., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bezstarosti, Bakker, Kramer, de Fijter, Reinders, Mulder, Claas and Heidt.)

HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study, we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively, while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs, multiple uniquely shared amino acid configurations were identified, warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes, which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation., Competing Interests: RB is employed by Pure Protein LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bezstarosti, Kramer, Franke-van Dijk, Vergunst, Bakker, Uyar-Mercankaya, Buchli, Roelen, de Fijter, Claas and Heidt.)