RSR RSR RSR RSR RSR. ElisaRSR AQP4 Ab RSR. Aquaporin-4 Autoantibody Assay Kit
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1 To aid diagnosis of Neuromyelitis Optica (NMO) and NMO spectrum disorder (NMOSD) To confirm diagnosis before initial treatment of patients with demyelinating inflammatory disease NMO, NMOSD and AQP4 Elisa AQP4 Ab 7,101,679B2, European patent , Japanese patent and related patents and patents pending in other countries To differentiate NMO from multiple sclerosis (MS) and different forms of transverse myelitis To monitor and follow-up NMOSD patients General Info What is NMO? Neuromyelitis optica (NMO), also known as Devic s syndrome, is an immune-mediated neurologic disease that affects the spinal cord and optic nerves. It can be considered to be a disorder distinct from multiple sclerosis (MS). In 2004, a serum immunoglobulin autoantibody (NMO-IgG) was shown to be a specific marker for NMO and subsequently the water channel aquaporin-4 (AQP4) was identified as the target antigen for NMO-IgG. What is AQP4? AQP4 is a water selective transporter in the plasma membrane. It is the most abundant water channel in the brain expressed in astrocytes and ependymal cells through out the brain and the spinal cord. It is involved in movement of water between blood and brain, and between brain and the CSF compartment. What is NMOSD? Neuromyelitis optica spectrum disorders (NMOSD) include recurrent optic neuritis (often severe and bilateral), longitudinally extensive transverse myelitis (LETM), and occasionally symptomatic encephalitis (uncommon in adults but encountered in 45% NMO-IgG seropositive children). Schematic structure of AQP4 Structure of human AQP4 Loop C Loop A Loop E outside AQP4 M23 membrane corresponds to MW inside 32kDa Loop B Loop D AQP4 M1 corresponds NH 2 to MW COOH 34kDa Limited 2010 Limited. All Rights Reserved 1
2 MS or NMO? What are MS and NMO? Elisa AQP4 Ab Multiple sclerosis is primarily an inflammatory disorder of the brain and spinal cord in which focal lymphocytic infiltration leads to damage of myelin and axons. MS and NMO have some overlapping manifestations but can be distinguished using clinical, radiological, prognostic and pathology indicators and by a serum biomarker, NMO-IgG ie aquaporin-4 autoantibody (AQP4 Ab). 7,101,679B2, European patent , Japanese patent and related patents and patents pending in other countries General Info Geographic distribution MS NMO Common: in Western Europe, North America, Common: in Asia, Caribbean and South America Australia and New Zealand Differences between MS and NMO Patients Clinical features Immunopathology MRI Laboratory tests Multiple sclerosis (MS) 70 % Female yrs Rare in non-white ancestry Not associated with autoimmunity Mild optic neuritis and myelitis Common and early brain involvement Relapsing, progressive clinical course Leukocyte infiltrates are mainly T and B lymphocytes Upregulation of aquaporin-4 in active lesions Less-marked complement deposits within macrophages and myelin sheath Peripheral, asymmetric lesions Usually ovoid periventricular lesions CSF oligoclonal bands present NMO-IgG negative Neuromyelitis optica (NMO) 90 % Female yrs Common with non-white ancestry Associated with autoimmunity Severe optic neuritis and myelitis Uncommon or late brain involvement Relapsing, rarely progressive clinical course Leukocyte infiltrates are mainly neutrophils and eosinophils Loss of aquaporin-4 in all lesions Complement and IgM/IgG deposits around blood vessels Longitudinally extensive central lesions Normal or nonspecific brain MRI CSF oligoclonal bands absent NMO-IgG positivity 70% Therapy Immunomodulation Immunosuppression Limited Adapted from Brion G Arch Neurology : Limited. All Rights Reserved 2
3 NMO-IgG vs Elisa TM AQP4 Ab Revised (2006) diagnostic criteria for neuromyelitis optica (NMO) Elisa AQP4 Ab NMO-IgG, identified as AQP4 Ab aids diagnosis of NMO and NMOSD 7,101,679B2, European patent , Japanese patent and related patents and patents pending in other countries Definite NMO Optic neuritis Acute myelitis Plus at least two out of three supportive criteria: 1) Contiguous spinal cord MRI lesion extending over three or more vertebral segments 2) Brain MRI not meeting diagnostic criteria for multiple sclerosis 3) NMO-IgG seropositive status Clinical Info Comparison: NMO-IgG by immunofluorescence assay vs AQP4 Ab by Elisa TM AQP4 Ab From Wingerchuk DM et al Neurology 2006;66: IFT titer n= NMO-IgG IFT Elisa TM AQP4 Ab Concordance rate: 94% Cut-off : Concentration of AQP4Ab (units/ml) NMO-IgG: detection of immunoglobulin (IgG) specific for NMO patients by indirect immunofluourescence (IFT) method using sections of normal mouse brain tissue AQP4 Ab: detection of specific AQP4 Ab using Elisa TM AQP4 Ab RESULTS: 61/62 (98%) IFT positive sera were positive for AQP4 Ab in Elisa TM AQP4 Ab. 5/34 (15%) IFT negative NMO patient sera were positive for AQP4 Ab in Elisa TM AQP4 Ab. Limited 2010 Limited. All Rights Reserved 3
4 Elisa TM AQP4 Ab Elisa AQP4 Ab 7,101,679B2, European patent , Japanese patent and related patents and patents pending in other countries Measurements in different patients groups (units/ml) Clinical Info Cut-off :5 0 NMO-IgG(+) NMO-IgG (-) HBD MS MG TRAb (+) GADAb (+) 21-OHAb (+) dsdna Ab(+) by IFT by IFT n = 216 n = 26 n =36 n = 102 n = 41 n = 27 n = 10 n = 62 n = 34 Samples from 96 NMO patients, 216 healthy blood donors, 26 multiple sclerosis (MS) patients and 216 patients with autoimmune diseases other than NMO were tested for AQP4 Ab using Elisa TM AQP4 Ab. RESULTS: 61/62 (98%) NMO-IgG positive by IFT sera were positive for AQP4 Ab, 5/34 (15%) NMO-IgG negative by IFT NMO patients sera were positive for AQP4 Ab. All 216 (100%) healthy controls were identified as being negative for AQP4 Ab. None of 26 MS patients, nor 216 patients with autoimmune diseases other than NMO except 1 patient (out of 102) with Graves disease (TRAb positive) and 1 patient (out of 36) with myasthenia gravis (MG) were positive for AQP4 Ab. Limited 2010 Limited. All Rights Reserved 4
5 Elisa TM AQP4 Ab Elisa AQP4 Ab 7,101,679B2, European patent , Japanese patent and related patents and patents pending in other countries Description Enzyme linked Immunosorbent assay (ELISA) kit for the quantitative determination of autoantibodies to aquaporin-4 (AQP4) in serum Assay principle + + AQP4 coated AQP4 Ab in AQP4 - Biotin well test sample Detect AQP4 Biotin bound by addition of streptavidin peroxidase and colorogenic substrate. Read OD at 450nm and 405nm. Kit performance Sensitivity: 98%, n = 62 of NMO-IgG positive sera (by IFT) Specificity: 100%, n = 216 of healthy blood donor sera Lower Detection 0.16 units/ml (mean + 2 standard Limit: deviations of negative control; n = 20) Calibrator range: units/ml Cut-off: Negative: <5 units/ml Positive: 5 units/ml Cross reactivity: None of 26 MS patients, nor 216 patients with autoimmune diseases other than NMO except 1 patient (out of 102) with Graves disease (TRAb positive) and 1 patient (out of 36) with myasthenia gravis (AChR Ab positive) were positive for AQP4 Ab. Inter assay Variation (n=20): Sample units/ml CV (%) Technical Info Intra assay Variation (n=25): Sample units/ml CV (%) Calibration curve Calibration curve and dilutions of AQP4 Ab positive samples Absorbance at 450nm Calibration curve 2 Patient Patient Patient Patient Patient Concentration of AQP4Ab (units/ml) Concentration of AQP4 Ab (units/ml) A calibration curve is established by plotting calibrator concentrations: on the x-axis (log scale) against the absorbance of the calibrators on the y-axis (linear scale). Limited Absorbance at 450nm The absorbance of diluted sera from NMO patient 1 (from 1:256 to 1:16384), patient 2 (neat to 1:16), patient 3 (1:2 to 1:32), patient 4 (neat to 1:16) and patient 5 (1:8 to 1:512) decreased in a dosedependent manner and followed closely the calibration curve Limited. All Rights Reserved 5
6 Elisa TM Assay procedure AQP4 Ab Elisa AQP4 Ab 7,101,679B2, European patent , Japanese patent and related patents and patents pending in other countries Assay plan (samples tested in duplicate) Total assay time: 3 hours Assay plan Volume Time Pipette: Calibrators, controls and patient samples 50 µl Pipette: AQP4-Biotin (reconstituted) 25 µl Incubate: On an ELISA plate shaker at 500 shakes /min Aspirate/decant and wash: x 3 Pipette: SA-POD (diluted 1:20) 100 µl Incubate: On an ELISA plate shaker at 500 shakes /min Aspirate/decant and wash: x 3 Pipette: TMB 100 µl 2 hrs 20 min Incubate: Without shaking in dark room 20 min Pipette: Stop solution 100 µl Shake: Read: Absorbance at 450nm and 405 nm 5 sec Wells required for controls: 16 wells (as below). Maximum of 40 samples on one ELISA plate. 3 separate assays can be run with 1 kit (96 wells), (sample number 8 per assay) A B C3 S1 S5 B B C3 S1 S5 C N C4 S2 S6 D N C4 S2 S6 E C1 PI S3 S7 F C1 PI S3 S7 G C2 PII S4 S8 H C2 PII S4 S8 B: blank PI II: positive controls I & II N: negative control S: patient samples C1-4: calibrators 5u/mL, 30u/mL, 75u/mL, 160 u/ml respectively Technical Info PRODUCTS - NEUROIMMUNOLOGY Disease group and product Type Kit size (tube/well) Order code Sample Total volume assay time / tube or well Cut-off Calibrator Range Sensitivity CE mark in EU Myasthenia Gravis (MG) 25 RBA/25 Ria TM AChR Ab RIA 50 RBA/50 5µL 5 hours Negative: <0.5 nmoles/l nmoles/l Positive: 0.5 nmoles/l 82% 100 RBA/100 Product Info Elisa TM AChR Ab ELISA 96 ACE/96 40µL 21 hours Negative: <0.45 nmoles/l nmoles/l 92% Positive: 0.45 nmoles/l Neuromyelitis Optica (NMO) and NMO spectrum disorder (NMOSD) Elisa TM AQP4 Ab ELISA 96 AQP4/96 50µL 3 hours Lambert-Eaton Myasthenic Syndrome (LEMS) Ria TM VGCC Ab RIA 25 LEMS/25 25µL 3 hours Autoimmune Voltage-Gated Potassium Channelopathies and related disorders Negative: <5 units/ml Positive: 5 units/ml Negative: 30 nmoles/l Positive: 30 nmoles/l units/ml N/A N/A N/A Ria TM VGKC Ab RIA 25 VGK/25 5µL 22 hours Limited Negative: <85 pmoles/l Positive: 85 pmoles/l N/A N/A - As of March Limited. All Rights Reserved 6
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