A Flow Cytometric Assay to Detect Functional Ganglionic Acetylcholine Receptor Antibodies by Immunomodulation in Autoimmune Autonomic Ganglionopathy
Autoimmune Autonomic Ganglionopathy (AAG) is an uncommon immune-mediated neurological disease that results in failure of autonomic function and is associated with autoantibodies directed against the ganglionic acetylcholine receptor (gnACHR). The antibodies are routinely detected by immunoprecipitat...
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Frontiers Media S.A.
2021-06-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2021.705292/full |
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| author | Nicolás Urriola Nicolás Urriola Judith M. Spies Judith M. Spies Katrina Blazek Katrina Blazek Bethan Lang Stephen Adelstein Stephen Adelstein Stephen Adelstein |
| author_facet | Nicolás Urriola Nicolás Urriola Judith M. Spies Judith M. Spies Katrina Blazek Katrina Blazek Bethan Lang Stephen Adelstein Stephen Adelstein Stephen Adelstein |
| author_sort | Nicolás Urriola |
| collection | DOAJ |
| description | Autoimmune Autonomic Ganglionopathy (AAG) is an uncommon immune-mediated neurological disease that results in failure of autonomic function and is associated with autoantibodies directed against the ganglionic acetylcholine receptor (gnACHR). The antibodies are routinely detected by immunoprecipitation assays, such as radioimmunoassays (RIA), although these assays do not detect all patients with AAG and may yield false positive results. Autoantibodies against the gnACHR exert pathology by receptor modulation. Flow cytometric analysis is able to determine if this has occurred, in contrast to the assays in current use that rely on immunoprecipitation. Here, we describe the first high-throughput, non-radioactive flow cytometric assay to determine autoantibody mediated gnACHR immunomodulation. Previously identified gnACHR antibody seronegative and seropositive sera samples (RIA confirmed) were blinded and obtained from the Oxford Neuroimmunology group along with samples collected locally from patients with or without AAG. All samples were assessed for the ability to cause gnACHR immunomodulation utilizing the prototypical gnACHR expressing cell line, IMR-32. Decision limits were calculated from healthy controls, and Receiver Operating Characteristic (ROC) curves were constructed after unblinding all samples. One hundred and ninety serum samples were analyzed; all 182 expected negative samples (from healthy controls, autonomic disorders not thought to be AAG, other neurological disorders without autonomic dysfunction and patients with Systemic Lupus Erythematosus) were negative for immunomodulation (<18%), as were the RIA negative AAG and unconfirmed AAG samples. All RIA positive samples displayed significant immunomodulation. There were no false positive or negative samples. There was perfect qualitative concordance as compared to RIA, with an Area Under ROC of 1. Detection of Immunomodulation by flow cytometry for the identification of gnACHR autoantibodies offers excellent concordance with the gnACHR antibody RIA, and overcomes many of the shortcomings of immunoprecipitation assays by directly measuring the pathological effects of these autoantibodies at the cellular level. Further work is needed to determine the correlation between the degree of immunomodulation and disease severity. |
| first_indexed | 2024-12-21T16:04:11Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-21T16:04:11Z |
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| series | Frontiers in Immunology |
| spelling | doaj.art-c3dcef18e0af4a129a03703ab6e661bc2022-12-21T18:57:56ZengFrontiers Media S.A.Frontiers in Immunology1664-32242021-06-011210.3389/fimmu.2021.705292705292A Flow Cytometric Assay to Detect Functional Ganglionic Acetylcholine Receptor Antibodies by Immunomodulation in Autoimmune Autonomic GanglionopathyNicolás Urriola0Nicolás Urriola1Judith M. Spies2Judith M. Spies3Katrina Blazek4Katrina Blazek5Bethan Lang6Stephen Adelstein7Stephen Adelstein8Stephen Adelstein9Department of Clinical Immunology and Allergy, Royal Prince Alfred Hospital, Sydney, NSW, AustraliaFaculty of Medicine and Health, The University of Sydney, Sydney, NSW, AustraliaFaculty of Medicine and Health, The University of Sydney, Sydney, NSW, AustraliaDepartment of Neurology, Royal Prince Alfred Hospital, Sydney, NSW, AustraliaFaculty of Medicine and Health, The University of Sydney, Sydney, NSW, AustraliaCentre for Kidney Research, Children’s Hospital at Westmead, Sydney, NSW, AustraliaNuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United KingdomDepartment of Clinical Immunology and Allergy, Royal Prince Alfred Hospital, Sydney, NSW, AustraliaFaculty of Medicine and Health, The University of Sydney, Sydney, NSW, AustraliaCentral Sydney Immunopathology Laboratory, NSW Health Pathology, Sydney, NSW, AustraliaAutoimmune Autonomic Ganglionopathy (AAG) is an uncommon immune-mediated neurological disease that results in failure of autonomic function and is associated with autoantibodies directed against the ganglionic acetylcholine receptor (gnACHR). The antibodies are routinely detected by immunoprecipitation assays, such as radioimmunoassays (RIA), although these assays do not detect all patients with AAG and may yield false positive results. Autoantibodies against the gnACHR exert pathology by receptor modulation. Flow cytometric analysis is able to determine if this has occurred, in contrast to the assays in current use that rely on immunoprecipitation. Here, we describe the first high-throughput, non-radioactive flow cytometric assay to determine autoantibody mediated gnACHR immunomodulation. Previously identified gnACHR antibody seronegative and seropositive sera samples (RIA confirmed) were blinded and obtained from the Oxford Neuroimmunology group along with samples collected locally from patients with or without AAG. All samples were assessed for the ability to cause gnACHR immunomodulation utilizing the prototypical gnACHR expressing cell line, IMR-32. Decision limits were calculated from healthy controls, and Receiver Operating Characteristic (ROC) curves were constructed after unblinding all samples. One hundred and ninety serum samples were analyzed; all 182 expected negative samples (from healthy controls, autonomic disorders not thought to be AAG, other neurological disorders without autonomic dysfunction and patients with Systemic Lupus Erythematosus) were negative for immunomodulation (<18%), as were the RIA negative AAG and unconfirmed AAG samples. All RIA positive samples displayed significant immunomodulation. There were no false positive or negative samples. There was perfect qualitative concordance as compared to RIA, with an Area Under ROC of 1. Detection of Immunomodulation by flow cytometry for the identification of gnACHR autoantibodies offers excellent concordance with the gnACHR antibody RIA, and overcomes many of the shortcomings of immunoprecipitation assays by directly measuring the pathological effects of these autoantibodies at the cellular level. Further work is needed to determine the correlation between the degree of immunomodulation and disease severity.https://www.frontiersin.org/articles/10.3389/fimmu.2021.705292/fullimmunoassayflow cytometry—methodsneuroimmunologyautoimmune autonomic ganglionopathydiagnostic test |
| spellingShingle | Nicolás Urriola Nicolás Urriola Judith M. Spies Judith M. Spies Katrina Blazek Katrina Blazek Bethan Lang Stephen Adelstein Stephen Adelstein Stephen Adelstein A Flow Cytometric Assay to Detect Functional Ganglionic Acetylcholine Receptor Antibodies by Immunomodulation in Autoimmune Autonomic Ganglionopathy Frontiers in Immunology immunoassay flow cytometry—methods neuroimmunology autoimmune autonomic ganglionopathy diagnostic test |
| title | A Flow Cytometric Assay to Detect Functional Ganglionic Acetylcholine Receptor Antibodies by Immunomodulation in Autoimmune Autonomic Ganglionopathy |
| title_full | A Flow Cytometric Assay to Detect Functional Ganglionic Acetylcholine Receptor Antibodies by Immunomodulation in Autoimmune Autonomic Ganglionopathy |
| title_fullStr | A Flow Cytometric Assay to Detect Functional Ganglionic Acetylcholine Receptor Antibodies by Immunomodulation in Autoimmune Autonomic Ganglionopathy |
| title_full_unstemmed | A Flow Cytometric Assay to Detect Functional Ganglionic Acetylcholine Receptor Antibodies by Immunomodulation in Autoimmune Autonomic Ganglionopathy |
| title_short | A Flow Cytometric Assay to Detect Functional Ganglionic Acetylcholine Receptor Antibodies by Immunomodulation in Autoimmune Autonomic Ganglionopathy |
| title_sort | flow cytometric assay to detect functional ganglionic acetylcholine receptor antibodies by immunomodulation in autoimmune autonomic ganglionopathy |
| topic | immunoassay flow cytometry—methods neuroimmunology autoimmune autonomic ganglionopathy diagnostic test |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2021.705292/full |
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