An Easy and Sensitive Method to Profile the Antibody Specificities of HLA–specific Memory B Cells

BACKGROUND. Pretransplant immunological risk assessment is currently based on donor–specific HLA antibodies in serum. Despite being an excellent source for antibodies produced by bone marrow–residing plasma cells, serum analysis does not provide information on the memory B–cell compartment. Although B–cell culture supernatants can be used to detect memory B cell–derived HLA antibodies, low IgG concentrations can preclude detectability of HLA antibodies in luminex single–antigen bead (SAB) assays. METHODS. Culture supernatants of polyclonally activated B cells from alloantigen exposed (n = 13) or nonexposed (n = 10) individuals were either concentrated 10–fold, or IgG was isolated by using a protein G affinity purification method to increase the IgG concentration. These processed culture supernatants, as well as paired serum samples were tested for the presence of HLA antibodies using luminex SAB analysis. RESULTS. In immunized individuals, 64% were found to have HLA–specific B–cell memory in concentrated supernatants, whereas 82% showed HLA–specific B–cell memory when IgG isolated supernatants were used for HLA antibody detection. IgG–isolated supernatants showed higher mean fluorescence intensity values compared with concentrated supernatants without increased background. In some individuals, HLA–specific B–cell memory was detected in the absence of accompanying serum antibody specificities. CONCLUSIONS. We developed a novel, highly sensitive method to assess the HLA–specific memory B–cell compartment using luminex SAB technology. This assay allows direct comparison to the serum compartment and may therefore provide a more complete picture of the humoral alloimmune response in patients with a history of alloantigen exposure.