TY - JOUR
T1 - Development of an aquaporin-4 orthogonal array of particle-based ELISA for neuromyelitis optica autoantibodies detection
AU - Pisani, Francesco
AU - Settanni, Paolo
AU - Rosito, Stefania
AU - Mola, Maria Grazia
AU - Iorio, Raffaele
AU - Tortorella, Carla
AU - Ruggieri, Maddalena
AU - Trojano, Maria
AU - Svelto, Maria
AU - Frigeri, Antonio
AU - Nicchia, Grazia Paola
PY - 2015/11/1
Y1 - 2015/11/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84958026068&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84958026068&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0143679
DO - 10.1371/journal.pone.0143679
M3 - Article
AN - SCOPUS:84958026068
VL - 10
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 11
M1 - e0143679
ER -