A Flow Cytometric Assay to Detect Functional Ganglionic Acetylcholine Receptor Antibodies by Immunomodulation in Autoimmune Autonomic Ganglionopathy.

Nicolás Urriola,Katrina Blazek,Stephen Adelstein,Bethan Lang,Judith M Spies

doi:10.3389/fimmu.2021.705292

Abstract

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.

Highlights

Autoimmune Autonomic Ganglionopathy (AAG) is a rare disorder where autoantibodies against alpha3 containing ganglionic acetylcholine receptors interrupt acetylcholine signaling in autonomic ganglia, most commonly leading to orthostatic hypotension, gastrointestinal dysmotility, degrees of anhidrosis [1, 2], and less commonly, abnormal or absent pupillary light reflexes, dry eyes and mouth, as well as dysfunction of genitourinary, cardiovagal and erectile systems [3].The gnACHR are present on the surface of neurones
There was complete qualitative congruence between results obtained by radioimmunoprecipitation assay (RIA) and the novel flow cytometric assay; all positive samples by RIA were positive by flow cytometry, and all samples from disease and Healthy Controls were negative by flow cytometry
This study demonstrates that immunomodulation of gnACHR on IMR-32 cells as determined by flow cytometry performs at least as well as RIA in the detection of pathogenic gnACHR antibodies (Figures 3, 4)

Summary

Introduction

Autoimmune Autonomic Ganglionopathy (AAG) is a rare disorder where autoantibodies against alpha containing ganglionic acetylcholine receptors (gnACHR) interrupt acetylcholine signaling in autonomic ganglia, most commonly leading to orthostatic hypotension, gastrointestinal dysmotility, degrees of anhidrosis [1, 2], and less commonly, abnormal or absent pupillary light reflexes, dry eyes and mouth, as well as dysfunction of genitourinary, cardiovagal and erectile systems [3].The gnACHR are present on the surface of neurones. Binding assays include the radioimmunoprecipitation assay (RIA) and the Luciferase Immunoprecipitation assay (LIPS), that are reported to have a sensitivity and specificity of 50 and 100%, respectively [3, 5], with antibody levels directly correlated with severity of autonomic impairment. The RIA is, only moderately specific (92%) in AAG with severe autonomic dysfunction, with low positive levels not associated with autonomic impairment [8]. Positive results can be seen in up to 16.3% of patients with autoimmune disorders without autonomic impairment when assessed by the LIPS assay [9]

Methods

Results

Conclusion