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

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.

Novel Cell-Based Assay for Alpha-3 Nicotinic Receptor Antibodies Detects Antibodies Exclusively in Autoimmune Autonomic Ganglionopathy

“… 5 , 6 In addition, another group 10 developed a luciferase immunoprecipitation system (LIPS) for the detection of antibodies to individually expressed α3 or β4 nAChR subunits. 10 Recently, a FACS assay has been described 25 with IMR32 cells, detecting potentially pathogenic α3-nAChR antibodies in high RIPA samples, in agreement with an earlier study on the immunomodulating capacity of the α3-nAChR antibodies. 26 This assay may prove an attractive diagnostic assay, especially if it can substitute the IMR32 cells with α3-nAChR–transfected and control-untransfected cells and confirm a high specificity and sensitivity.…”

Autoantibodies against gAChR in humans and mouse models of autoimmune autonomic ganglionopathy

“…Using a flow cytometric gating strategy, Urriola et al quantified autoantibody-induced gAChR internalization. 15 All positive samples in radioimmunoassays demonstrated significant immunomodulation, and there were no false-positive or -negative samples using this method. These researchers described the first high-throughput, nonradioactive flow cytometric assay to determine autoantibody-mediated gAChR immunomodulation.…”

“…Given that the gAChR is expressed on the cell surface, it may be ideal to identify disease‐associated gAChR antibodies using cell‐based assays (CBAs), which can provide conformational epitopes for autoantibody binding. Using a flow cytometric gating strategy, Urriola et al quantified autoantibody‐induced gAChR internalization 15 . All positive samples in radioimmunoassays demonstrated significant immunomodulation, and there were no false‐positive or ‐negative samples using this method.…”

Effectiveness of treatment for 31 patients with seropositive autoimmune autonomic ganglionopathy in Japan

“…Finally, important differences were found in the antibody detection methods used. Although radioimmunoprecipitation assays in the United States, 1 – 3 LIPS assays in Japan, 4 , 14 and flow cytometric assays in Australia 25 , 26 are commonly used to detect gAChR antibodies, the sensitivity and specificity of each method vary, and validation studies should be performed. In the future, a multicenter prospective randomized, placebo-controlled study within an international framework is warranted to overcome these limitations to standardize treatment of patients with AAG.…”