OR45 Proof of principle: Bead-based ABO antibody assessment

Aim ABO antibody (ABO-Ab) titration is performed for ABO-incompatible (ABOi) transplantation management and is traditionally assessed by hemagglutination. However, hemagglutination cannot readily differentiate Ab isotype, nor distinguish between Abs to ABO-subtype antigens, which are expressed differently on organs vs erythrocytes. Our goal was to explore Luminex as a potential platform for the detection of IgG and IgM antibodies to ABO subtype antigens. Methods Phase I: Human blood group antigen subtype A-II was synthesized and conjugated to bovine serum albumin (BSA). BSA-A-II conjugates were coupled to Luminex beads. Coupling was assessed using phycoerythrin (PE)-labeled mouse monoclonal IgG and IgM anti-human ABO-A. Increasing BSA-A-II antigen concentrations were tested to optimize MFI. A Luminex-200 was used for bead acquisition. Phase II: Antigens ABO A-III and A-IV were similarly tested (alone and multiplexed) using additional Luminex beads. Phase III: Dilutions of PE-labeled anti-human IgG conjugate were tested against dilutions of anti-ABO-A plasma (blood group B donor) in a checkerboard titration. BSA-A-II coupled beads were used. Results Bead coupling was optimal at 5ug of BSA-A-II antigen. The coupling of BSA-A-II, -III and -IV was successful as demonstrated by a linear increase in MFI with increasing concentration of primary antibody (Figure). Beads were successfully labeled with monoclonal ABO-Ab when multiplexed. The assay appears most sensitive with the lowest background when patient plasma is diluted 1/25. Conclusions HLA laboratories are uniquely situated to assess ABO antibodies by Luminex as expertise and instrumentation already exist. This novel assay has the potential to be adopted by clinical HLA laboratories for rapid, specific, and sensitive assessment of IgM/IgG ABO subtype-specific ABO antibodies in ABOi transplantation. We continue to optimize the assay, include additional controls, and assess additional ABO subtypes. We are also testing flow-based bead platforms. Download high-res image (185KB) Download full-size image