A Novel Method for Anti-HLA Antibody Detection Using Personalized Peptide Arrays

BACKGROUND: HLA mismatches are the primary cause of alloantibody-mediated rejection (AMR) in organ transplantation. To delineate antigenic and immunogenic potentials among individual HLA mismatches, information regarding antibody specificity at the epitope level, instead of the allelic level, is needed. METHODS: This study explores a direct screening method for HLA linear epitopes in kidney transplant patients. We custom synthesized a large panel of 15-residue HLA peptides in an array format and measured alloantibody reactivity to these peptides from the sera of post and/or pretransplant patients. Two design concepts for the arrays were followed: a standard array of a fixed panel of peptides or personalized arrays. The standard array contains 420 peptides derived from a predetermined set of HLA-DQ allelic antigens based on templates also used in the single-antigen beads assay. RESULTS: The array detected distinct antiserum patterns among transplant subjects and revealed epitope levels of specificity largely in accordance with the single-antigen results. Two personalized arrays that each included donor-derived peptides of HLA-A, -B, -C, -DQ, and -DR sequences were separately designed for 2 transplant subjects. The personalized arrays detected de novo antibodies following transplantation. The new method also showed superior sensitivity to a single-antigen assay in one of the cases whose pathological diagnosis of AMR occurred before single-antigen assay could detect antibodies. CONCLUSIONS: This pilot study proved the feasibility of using personalized peptide arrays to achieve detection of alloantibodies for linear HLA epitopes associated with distinct donor-recipient mismatches. Single or multiple reactive epitopes may occur on an individual HLA molecule, and donor-specific HLA-DQ-reactivity among 5 kidney transplant subjects revealed patterns of shared epitopes.