180-P

Abstract

Aim Single antigen bead (SAB) assay allows in-depth characterization of HLA antibody specificities. In addition to detection of antibodies against natural HLA antigens, the SAB may also detect antibodies against denatured HLA antigens. Recently, we observed antibody reactivity to all DR antigens (pan-DR) in serum samples of two transplant candidates using the Luminex SAB assay. This study was to characterize the pan-DR antibodies detected by Luminex SAB on two serum samples. Methods Serum samples received from two transplant candidates were analyzed in the study. Initial antibody screening and specificity tests were performed using Flow PRA and Luminex SAB. Additional tests using Flow SAB, Flow phenotype bead assay and Flow T and B cell crossmatch were performed to confirm the presence of HLA antibodies. Results Flow PRA was clearly negative for both samples, while Luminex SAB surprisingly revealed antibody reactivity to all DR antigens. No antibodies to DQ and DP were detected. However, no HLA antibodies were detected by Flow phenotype bead assay. In the Flow SAB assay, patient #1 showed reactivity with majority of the DR antigen beads, while patient #2 showed no any binding reactivity. The crossmatch with a donor cell carrying DR4, DR15, DR51, and DR53 was completely negative for both serum samples. No specific antibody removals were observed on serum samples adsorbed using the donor cells in a Luminex SAB assay. Conclusions Flow PRA and Flow phenotype beads are coated with natural HLA antigens extracted from cell lines, while SABs are coated with recombinant HLA antigens. The antibody binding detected by SAB, but not by FlowPRA and Flow phenotype, may indicate the presence of antibodies against denatured antigens. Our results suggest that the pan-DR antibodies detected in these patients were possibly antibodies against denatured HLA antigens, more likely to a shared epitope uncovered on denatured DR antigens. Antibodies to denatured antigens would not result in crossmatch reactivity.