Identification of HLA-DR linear antibody epitopes in transplant patient sera using a multiplexed peptide immunoassay

Molecular mismatch analysis is increasingly used to assess donor-recipient compatibility in transplantation. We sought to determine if reactivity against continuous HLA-DR epitopes could be directly measured using a peptide-based immunoassay. We used sequence and informatics analysis to identify putative short linear epitopes in two polymorphic regions surrounding amino acid (aa) position 30 and 70 of HLA-DRB1*07:01 and HLA-DRB1*08:01, respectively. We tested sera from patients who were HLA antibody negative, HLA class II antibody positive for irrelevant antigens, or positive for the HLA-DR7 or -DR8 antigen by Single Antigen Bead testing. Among HLA-DR7 reactive sera, we found that 9/18 (50%) were above a positive threshold for one or both peptide epitopes, while 7/19 (36%) sera were positive for at least one HLA-DR8 peptide epitopes. This proof-of-concept study demonstrates that short peptide targets can be used to identify HLA antibody reactivity, which could be used to directly interrogate HLA epitope-level humoral alloimmunity.