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Strategy for anti-aquaporin-4 auto-antibody identification and quantification using indirect immunofluorescence and a new cell-based assay

In 2004, a highly disease-specific autoantibody named NMO-IgG was discovered in patients with neuromyelitis optica (NMO) and NMO related diseases (i.e. relapsing optic neuritis and longitudinally extensive transverse myelitis). The target antigen of NMO-IgG was identified as aquaporin-4 (AQP4), the main water channel protein in the central nervous system (CNS). Pathogenicity of NMO-IgG is now well recognized and anti-AQP-4 antibodies (Abs) are used as a biomarker for the diagnosis and follow-up of NMO patients. The current gold standard for NMO-IgG identification is still indirect immunofluorescence (IIF) on sections of CNS tissues but several AQP-4-specific assays have been developed in order to specifically identify and quantify anti-AQP-4 Abs. Our aim in this study was to compare IIF and a new cell based assay (CBA) in NMO-IgG assessment and to propose a strategy for anti-AQP-4 Ab identification and quantification. We evaluated samples from 17 NMO-IgG positive NMO, 29 NMO-IgG negative NMO, 16 multiple sclerosis (MS), 9 clinically isolated syndromes (CIS) and 5 healthy controls. IIF was performed on primate cerebellum slides and we developed a new CBA for anti-AQP-4 Ab quantification by flow cytometry using HEK-293 AQP-4 overexpressing cells. We observed an excellent correlation between IIF and our CBA. Indeed, all the sera positive by IIF were confirmed with the CBA. At the opposite, all the control sera as well as the MS/CIS and the seronegative NMO patients (NMO-IgG negative by IIF) were negative by flow cytometry. Of note, even if serial dilution of an AQP-4 positive serum indicated that the CBA is more sensitive than IIF, none of the sera negative by IIF at the diagnosis were positive with the CBA. Interestingly, one NMO serum that exhibited an atypical punctuated staining around CNS microvessels by IIF was negative by flow cytometry suggesting that this patient presented with a demyelinating disease associated with a blood-brain barrier (BBB)-directed auto-Ab other than anti-AQP4. In conclusion, our study highlights the complementarity between IIF and our CBA in NMO patients. We propose to use IIF for the screening at diagnosis in order to identify auto-Abs targeting the BBB. A highly sensitive, AQP-4 specific and quantitative assay such as this CBA could be used thereafter to specifically identify the target of the Ab and to monitor its serum concentration under treatment.

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