Development of an Aquaporin-4 Orthogonal Array of Particle-Based ELISA for Neuromyelitis Optica Autoantibodies DetectionReport as inadecuate




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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.



Author: Francesco Pisani, Paolo Settanni, Stefania Rosito, Maria Grazia Mola, Raffaele Iorio, Carla Tortorella, Maddalena Ruggieri, Maria

Source: http://plos.srce.hr/



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