Nanodisc‐Based Platform for HLA Antibody Identification

ABSTRACT The presence of donor‐specific antibodies (DSAs) is strongly associated with antibody‐mediated rejection (AMR) and worse allograft outcomes. The current gold standard method for detecting anti‐human leukocyte antigens (HLA) antibodies is based on the binding to HLA molecules, which are immobilized on beads. However, this assay may lead to the modification of the tertiary structure of the molecules and exposure of cryptic epitopes, thus resulting in aberrant false‐positive and false‐negative reactivity. With the increasing reliance on virtual crossmatching (comparing anti‐HLA antibodies identified using the single antigen bead (SAB) assay to the donor HLA typing to identify DSAs) instead of a physical crossmatch, native and full‐length HLA proteins are critical for accurate HLA antibody identification. In this study, we incorporate native and full‐length HLA into phospholipid bilayer nanodiscs to form stable HLA‐nanodisc (HLA‐ND) complexes in a native‐like membrane environment that maintains structure and prevents denaturation for anti‐HLA antibody identification. Using these HLA‐ND complexes immobilized on beads, we show successful DSA identification with serum from kidney transplant recipients. Furthermore, we introduce a novel bioluminescence‐based bead‐free homogeneous immunoassay for detecting anti‐HLA antibodies using HLA‐ND complexes. This platform has the potential to accurately detect multiple anti‐HLA antibodies before and after solid organ transplantation.