title: Development of an Aquaporin-4 Orthogonal Array of Particle-Based ELISA for Neuromyelitis Optica Autoantibodies Detection.
abstract: 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.
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Pisani
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Settanni
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Rosito
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Mola
authors.affiliation: Department of Geriatrics, Neuroscience and Orthopedics, Institute of Neurology, Catholic University, Rome, Italy.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Iorio
authors.affiliation: Department of Basic Medical Sciences, Neuroscience and Sense Organs, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Tortorella
authors.affiliation: Department of Basic Medical Sciences, Neuroscience and Sense Organs, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Maddalena
authors.initials: M
authors.lastname: Ruggieri
authors.affiliation: Department of Basic Medical Sciences, Neuroscience and Sense Organs, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Trojano
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Svelto
authors.affiliation: Department of Basic Medical Sciences, Neuroscience and Sense Organs, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Frigeri
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Grazia Paola
authors.initials: GP
authors.lastname: Nicchia
mesh_terms: D000818:Animals; D051401:Aquaporin 4 / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D009471:Neuromyelitis Optica / Q000276:immunology*; D051381:Rats
chemical_list: D051401:Aquaporin 4; D001323:Autoantibodies; D007074:Immunoglobulin G