Development of an aquaporin-4 orthogonal array of particle-based ELISA for neuromyelitis optica autoantibodies detection

Serological markers of Nuromyelitis Optica (NMO), an autoimmune disorder of the central nervous system, are autoantibodies targeting the astrocytic water channel aquaporin-4 (AQP4). We have previously demonstrated that the main epitopes for these autoantibodies (AQP4-IgG) are generated by the supramolecular arrangement of AQP4 tetramers into an Orthogonal Array of Particles (OAPs). Many tests have been developed to detect AQP4- IgG in patient sera but several procedural issues affect OAP assembly and consequently test sensitivity. To date, the protein based ELISA test shows the lowest sensitivity while representing a valid alternative to the more sensitive cell based assay (CBA), which, however, shows economic, technical and interpretation problems. Here we have developed a high perfomance ELISA in which native OAPs are used as the molecular target. To this aim a native size exclusion chromatography method has been developed to isolate integral, highly pure and AQP4-IgG-recognized OAPs from rat brain. These OAPs were immobilized and oriented on a plastic plate by a sandwich approach and 139 human sera were tested, including 67 sera from NMO patients. The OAP-ELISA showed a 99% specificity and a higher sensitivity (91%) compared to the CBA test. A comparative analysis revealed an end-point titer three orders of magnitude higher than the commercial ELISA and six times higher than our in-house CBA test. We show that CNS-extracted OAPs are crucial elements in order to perform an efficient AQP4-IgG test and the OAP-ELISA developed represents a valid alternative to the CBA currently used. © 2015 Pisani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.