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Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-rAb), together with a C-terminus anti-AQP4 antibody. NMO-rAb titrations showed binding with dissociation constants down to 44 ? 7 nM. Different NMO-rAbs and NMO patient sera showed wide variation in NMO-IgG binding to M1 vs. M23 AQP4. Differences in binding affinity rather than stoichiometry accounted for M1 vs. M23 binding specificity, with consistently greater affinity of NMO-IgG binding to M23 than M1 AQP4. Binding and OAP measurements in cells expressing different M1:M23 ratios or AQP4 mutants indicated that the differential binding of NMO-IgG to M1 vs. M23 was due to OAP assembly rather than to differences in the M1 vs. M23 N-termini. Purified Fab fragments of NMO-IgG showed similar patterns of AQP4 isoform binding, indicating that structural changes in the AQP4 epitope upon array assembly, and not bivalent crosslinking of whole IgG, results in the greater binding affinity to OAPs. Our study establishes a quantitative assay of NMO-IgG binding to AQP4, and indicates remarkable, OAP-dependent heterogeneity in NMO autoantibody binding specificity.

Read More: BINDING AFFINITY AND SPECIFICITY OF NEUROMYELITIS OPTICA AUTOANTIBODIES TO AQUAPORIN-4 M1/M23 ISOFORMS AND ORTHOGONAL ARRAYS