OR34 Modernizing ABO antibody detection: Development of a bead-based ABO antibody detection assay

Abstract

Aim Accurate, clinically relevant characterization of ABO antibodies (ABO-Ab) is critical to assess their impact in ABO incompatible 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 a slide micro-array 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 control beads, respectively. Optimal plasma and anti-human IgG and IgM labelling-antibody dilutions were determined. IgG and IgM isotypes with specificities for ABO A-subtypes were measured (n = 40 healthy donors) by mean fluorescent intensity (MFI). Levels of IgG vsIgM ABO-A-Ab were compared (Wilcoxon signed-rank test). Preliminary positive MFI thresholds for each ABO A-subtype bead were calculated based on ABO-A-Ab in ABO A control plasma. Results Variation in ABO-Ab levels between ABO A-subtypes was detected. There was no significant difference between IgG and IgM ABO-Ab levels in paired MFI for all ABO types. IgG and IgM ABO-Ab are clearly detectable in all non-ABO A controls. Calculated MFI thresholds accurately interpreted positive ABO-A-Ab in ABO O samples for the IgG assay and most beads in the IgM assay. ABO B controls had similar IgM ABO-A-Ab levels to ABO O controls but lower levels of IgG. Reactivity to alpha-Gal was present but inconsistent. Conclusions This bead-based method successfully measures ABO-A-Ab and shows promise for clinical laboratory implementation. The specificity of these solid-phase assays will facilitate accurate assessment of ABO-Ab to subtypes, which are known to be expressed differently in endothelium than on erythrocytes. The ability to accurately measure both IgM and IgG ABO antibodies makes it possible to evaluate of the role of each ABO-Ab isotype in transplantation; this may be particularly relevant in the setting of plasmapheresis, which more efficiently removes IgM antibodies. Further optimization of this assay, controls, and ABO B and ABO H panel development are underway.