Detection of Acetylcholine Receptor Modulating Antibodies by Flow Cytometry

Lozier, Bucky K.; Haven, Thomas R.; Astill, Mark E.; Hill, Harry R.

doi:10.1309/ajcpyeor6sge8zlu

Cited by 20 publications

(14 citation statements)

References 11 publications

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“…Acetylcholine receptor (AChR) modulating antibody can be used to distinguish between patients with myasthenia gravis and those without myasthenia gravis who are AChR binding antibody positive. 10 In addition, patients with IgG that robustly modulates AChR (80% antigen loss or more) commonly have thymoma. 6 GlyRα1 is abundant in the retina and cerebrum, as well as brainstem and spinal cord.…”

Section: Discussionmentioning

confidence: 99%

Glycine receptor modulating antibody predicting treatable stiff-person spectrum disorders

Hinson

López-Chiriboga

Bower

et al. 2018

Neurol Neuroimmunol Neuroinflamm

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BackgroundGlycine receptor alpha-1 subunit (GlyRα1)-immunoglobulin G (IgG) is diagnostic of stiff-person syndrome (SPS) spectrum but has been reported detectable in other neurologic diseases for which significance is less certain.MethodsTo assess GlyRα1-IgGs as biomarkers of SPS spectrum among patients and controls, specimens were tested using cell-based assays (binding [4°C] and modulating [antigen endocytosing, 37°C]). Medical records of seropositive patients were reviewed.ResultsGlyRα1-IgG (binding antibody) was detected in 21 of 247 patients with suspected SPS spectrum (8.5%) and in 8 of 190 healthy subject sera (4%) but not CSF. Among 21 seropositive patients, 20 had confirmed SPS spectrum clinically, but 1 was later determined to have a functional neurologic disorder. Sera from 9 patients with SPS spectrum , but not 7 controls, nor the functional patient, caused GlyRα1 modulation (100% specificity). SPS spectrum phenotypes included progressive encephalomyelitis with rigidity and myoclonus (PERM) (8), classic SPS (5), stiff limb (5), stiff trunk (1), and isolated exaggerated startle (hyperekplexia, 1). Neuropsychiatric symptoms present in 12 patients (60%) were anxiety (11), depression (6), and delirium (3). Anxiety was particularly severe in 3 patients with PERM. Objective improvements in SPS neurologic symptoms were recorded in 16 of 18 patients who received first-line immunotherapy (89%, 9/10 treated with corticosteroids, 8/10 treated with IVIg, 3/4 treated with plasma exchange, and 1 treated with rituximab). Treatment-sparing maintenance strategies were successful in 4 of 7 patients (rituximab [2/3], azathioprine [1/1], and mycophenolate [1/3]).ConclusionsGlyRα1-modulating antibody improves diagnostic specificity for immunologically treatable SPS spectrum disorders.Classification of evidenceThis study provides Class IV evidence that GlyRα1-modulating antibody accurately identifies patients with treatable SPS spectrum disorders.

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Section: Discussionmentioning

confidence: 99%

Glycine receptor modulating antibody predicting treatable stiff-person spectrum disorders

Hinson

López-Chiriboga

Bower

et al. 2018

Neurol Neuroimmunol Neuroinflamm

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“…Expi 293, purchased from Thermo Fisher Scientific, was used to express recombinant Abs. The human rhabdomyosarcoma cell line TE671, which expresses human nAChR on its surface [ 29 , 30 ], and DB40, transfected with δ-subunit genes to CN21 (cells transfected with the ε-subunit of TE671) for the stable expression of both fetal and adult AChRs [ 31 , 32 ], were purchased from the American Type Culture Collection (ATCC) and ISIS Innovation Ltd., Oxford, respectively. These cell lines were used for in vitro assays, including the binding, AlphaLISA, downmodulation, antagonist/agonist, and competition assays.…”

Section: Methodsmentioning

confidence: 99%

“…Downmodulation of nAChRs on the surface of DB40 cells was monitored based on a method described by Lozier BK et al [ 31 ] with some modifications, particularly in detection. DB40 cells were detached with non-enzymatic dissociation solution (Sigma-Aldrich) from the culture flask and 5× 10 4 cells/well were resuspended in 200 μL of DMEM with 10% FBS in V-shaped 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

Analysis of peripheral B cells and autoantibodies against the anti-nicotinic acetylcholine receptor derived from patients with myasthenia gravis using single-cell manipulation tools

Makino¹,

Nakamura

Terakawa

et al. 2017

PLoS ONE

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The majority of patients with myasthenia gravis (MG), an organ-specific autoimmune disease, harbor autoantibodies that attack the nicotinic acetylcholine receptor (nAChR-Abs) at the neuromuscular junction of skeletal muscles, resulting in muscle weakness. Single cell manipulation technologies coupled with genetic engineering are very powerful tools to examine T cell and B cell repertoires and the dynamics of adaptive immunity. These tools have been utilized to develop mAbs in parallel with hybridomas, phage display technologies and B-cell immortalization. By applying a single cell technology and novel high-throughput cell-based binding assays, we identified peripheral B cells that produce pathogenic nAChRAbs in patients with MG. Although anti-nAChR antibodies produced by individual peripheral B cells generally exhibited low binding affinity for the α-subunit of the nAChR and great sequence diversity, a small fraction of these antibodies bound with high affinity to nativestructured nAChRs on cell surfaces. B12L, one such Ab isolated here, competed with a rat Ab (mAb35) for binding to the human nAChR and thus considered to recognize the main immunogenic region (MIR). By evaluating the Ab in in vitro cell-based assays and an in vivo rat passive transfer model, B12L was found to act as a pathogenic Ab in rodents and presumably in humans.These findings suggest that B cells in peripheral blood may impact MG pathogenicity. Our methodology can be applied not only to validate pathogenic Abs as molecular target of MG treatment, but also to discover and analyze Ab production systems in other human diseases.

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“…The ACh signal performs various biological functions, such as regulating physiological levels of different neurotransmitters, opening of ligand-gated ion channels, and bursting mode of neuronal firing. In hydrolysis and diffusion at synapses, imbalance of ACh is associated with several neurological and physiological diseases, such as Alzheimer’s [160] and myasthenia gravis [161]. In addition, levels of ACh change in behavioral, learning, and sleep disorders [162].…”

Section: Enzyme Sensorsmentioning

confidence: 99%

Latest Trends in Electrochemical Sensors for Neurotransmitters: A Review

Tavakolian-Ardakani

Hosu

Cristea

et al. 2019

Sensors

109

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Neurotransmitters are endogenous chemical messengers which play an important role in many of the brain functions, abnormal levels being correlated with physical, psychotic and neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's disease. Therefore, their sensitive and robust detection is of great clinical significance. Electrochemical methods have been intensively used in the last decades for neurotransmitter detection, outclassing more complicated analytical techniques such as conventional spectrophotometry, chromatography, fluorescence, flow injection, and capillary electrophoresis. In this manuscript, the most successful and promising electrochemical enzyme-free and enzymatic sensors for neurotransmitter detection are reviewed. Focusing on the activity of worldwide researchers mainly during the last ten years (2010–2019), without pretending to be exhaustive, we present an overview of the progress made in sensing strategies during this time. Particular emphasis is placed on nanostructured-based sensors, which show a substantial improvement of the analytical performances. This review also examines the progress made in biosensors for neurotransmitter measurements in vitro, in vivo and ex vivo.

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Detection of Acetylcholine Receptor Modulating Antibodies by Flow Cytometry

Abstract: The new flow cytometric method using the RD cell platform provided higher quality clinical results, a more robust and efficient assay format, a significant cost savings, and less environmental burden.

Cited by 20 publications

References 11 publications

Glycine receptor modulating antibody predicting treatable stiff-person spectrum disorders

Glycine receptor modulating antibody predicting treatable stiff-person spectrum disorders

Analysis of peripheral B cells and autoantibodies against the anti-nicotinic acetylcholine receptor derived from patients with myasthenia gravis using single-cell manipulation tools

Latest Trends in Electrochemical Sensors for Neurotransmitters: A Review

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