Solid-phase binding assays using purified human leukocyte antigen (HLA) allow the accurate identification and characterization of HLA-specific antibodies in organ transplant patients, but sera may contain factors that block the detection of clinically relevant alloantibodies. The effect of treating patient sera with dithiothreitol (DTT) on alloantibody detection was examined. In 49 sera submitted for routine HLA-specific antibody monitoring, DTT made little difference to immunoglobulin G HLA-specific antibody detection using LABScreen HLA class I and class II mixed beads. However, in sera submitted for antibody identification using single antigen beads (SAB), DTT markedly increased (>twofold increased median fluorescence intensity) antibody binding to HLA class I and/or class II specificities in 14 of 76 (18%) patient sera. In a cohort of highly sensitized patients, treatment of sera with DTT enhanced antibody-binding levels in 14 of 15 (93%) sera. This study highlights the need to consider routine testing of sera with and without DTT for analysis of HLA-specific antibodies by SAB.