*Purpose: Accurate characterization of ABO antibodies (ABO-Ab) is critical to assess their impact in ABO incompatible (ABOi) transplantation. The current ABO-Ab detection method using erythrocyte agglutination is limited by lack of ABO-subtype specificity, difficulty in ABO-Ab isotype differentiation, and poor reproducibility. We previously developed an ABO glycan microarray method for ABO-Ab analysis to address these limitations. Our aim was to create a similar bead solid phase assay.

*Methods: ABO A-subtype antigens (I,II,III,IV,V,VI) were coupled to Luminex beads and quantified using monoclonal ABO-Ab. Bovine serum albumin and alpha-Gal antigen were coupled as negative/positive controls, respectively. IgG and IgM isotypes with specificities for ABO A-subtypes were measured and compared (n=39 healthy adult donors) by mean fluorescence intensity (MFI). These samples were tested in parallel on the glycan array. Wilcoxon signed-rank test was used.

*Results: ABO-A- subtype specific antibodies were detected; there was a high degree of variability in the MFI values between subjects. IgG and IgM ABO-A-Ab were detectable in all non-ABO A subjects although some subjects demonstrated low MFI values by both bead and array (Figure 1A). ABO-B controls had similar IgM ABO-A-Ab levels to ABO O controls but lower levels of IgG. Also shown in Fig 1A, IgM ABO-Ab were detected at higher MFI in the bead vs array method for many subtypes. IgG vs IgM antibodies were compared (Figure 1B); the level of antibody of one isotype does not appear to predict the level of the other.

*Conclusions: This method successfully measures ABO-A-Ab and shows promise for clinical laboratory implementation, where bead-based assays are already used. A flow cytometry bead panel is in development with similar potential. The specificity of this assay will facilitate assessment of ABO-Ab to subtypes, which are known to be expressed differently in cardiac endothelium than on erythrocytes. The ability to measure both IgM and IgG ABO antibodies makes it possible to evaluate of the role of each isotype in transplantation; isotype ABO-Ab differentiation may be particularly relevant in the setting of plasmapheresis, which more efficiently removes IgM antibodies. There is also potential for use in ABOi renal and stem cell transplantation.

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