Multicentre comparison of a diagnostic assay: Aquaporin-4 antibodies in neuromyelitis optica

Objective Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). Methods Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). Results Results of tests on 92 controls identified 12assays as highly specific (0-1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5-100%) of all 21 assays. The specificities (85.8-100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. Conclusions The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology

Evaluation of a Multiparametric Immunofluorescence Assay for Standardization of Neuromyelitis Optica Serology

Background: Neuromyelitis optica (NMO) is a severely disabling autoimmune disorder of the central nervous system, which predominantly affects the optic nerves and spinal cord. In a majority of cases, NMO is associated with antibodies to aquaporin-4 (AQP4) (termed NMO-IgG). Aims: In this study, we evaluated a new multiparametric indirect immunofluorescence (IIF) assay for NMO serology. Methods: Sera from 20 patients with NMO, 41 patients with multiple sclerosis (MS), 30 healthy subjects, and a commercial anti-AQP4 IgG antibody were tested in a commercial composite immunofluorescence assay ("Neurology Mosaic 17"; Euroimmun, Germany), consisting of five different diagnostic substrates (HEK cells transfected with AQP4, non-transfected HEK cells, primate cerebellum, cerebrum, and optic nerve tissue sections). Results: We identified AQP4 specific and non-specific fluorescence staining patterns and established positivity criteria. Based on these criteria, this kit yielded a high sensitivity (95%) and specificity (100%) for NMO and had a significant positive and negative likelihood ratio (LR+ = ∞, LR- = 0.05). Moreover, a 100% inter- and intra-laboratory reproducibility was found. Conclusions: The biochip mosaic assay tested in this study is a powerful tool for NMO serology, fast to perform, highly sensitive and specific for NMO, reproducible, and suitable for inter-laboratory standardization as required for multi-centre clinical trials

Monocyte NOTCH2 expression predicts IFN-beta immunogenicity in multiple sclerosis patients

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-beta is an established treatment for MS; however, up to 30% of IFN-beta-treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14(+) monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-beta. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-beta administration. Reduced monocyte NOTCH2 expression in nADA(+) MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA(+) patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-beta administration

Detection and kinetics of persistent neutralizing anti-interferon-beta antibodies in patients with multiple sclerosis. Results from the ABIRISK prospective cohort study

Two validated assays, a bridging ELISA and a luciferase-based bioassay, were compared for detection of anti-drug antibodies (ADA) against interferon-beta (IFN-beta) in patients with multiple sclerosis. Serum samples were tested from patients enrolled in a prospective study of 18 months. In contrast to the ELISA, when IFN-beta-specific rabbit polyclonal and human monoclonal antibodies were tested, the bioassay was the more sensitive to detect IFN-beta ADA in patients' sera. For clinical samples, selection of method of ELISA should be evaluated prior to the use of a multi-tiered approach. A titer threshold value is reported that may be used as a predictor for persistently positive neutralizing ADA

Clinicogenomic factors of biotherapy immunogenicity in autoimmune disease: A prospective multicohort study of the ABIRISK consortium

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