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.

Keywords: autoimmune autonomic ganglionopathy; diagnostic test; flow cytometry—methods; immunoassay; neuroimmunology.

Figure 1

Pathology of autoantibody-induced cell surface receptor crosslinking and internalization (immunomodulation). The remaining surface receptors can be comparatively enumerated flow cytometrically. PE, phycoerythrin.

Figure 2

Flow cytometric gating strategy to quantify autoantibody induced ganglionic acetylcholine receptor (gnACHR) internalization. Cells are gated on live events (FVS660-low, through the APC channel), then subgated on small ‘neuroblast’ events with doublets excluded. The amount of gnACHR remaining on the surface of cells after sample serum is added is quantified by the amount of mab35 (followed by a PE-conjugated anti-rat IgG antibody) staining evident. (Sample A) incubated with Fetal Calf Serum (FCS), and only stained with a secondary antibody (unstained cells). Flow plots (middle of figure) display the percent of events positive for gnACHR as compared to the unstained cells. Histograms (to the right) display the same data. (Samples B–D) incubated with serum and stained with both primary and secondary antibodies. (B) = FCS (maximally stained cells), (C) = (typical) healthy control serum, (D) = serum from a patient with confirmed seropositive Autoimmune Autonomic Ganglionopathy.

Figure 3

Flow cytometric gnACHR modulation by patient autoantibodies. (A) Immunomodulation at screening dilution of 1:20. (B) Endpoint Titer of results (results with <18% immunomodulation being considered “Not Detected”). (C – inset) Receiver-Operator Characteristic (ROC) curve for all radioimmunoassay confirmed seropositive AAG serum samples, compared to all expected negative samples (Healthy Controls, Autonomic Disorders, Other Neurological Disorders and Systemic Lupus Erythematosus). Aurea Under ROC (AUROC) = 1).

Figure 4

(A) Correlation of flow cytometric-based immunomodulation assay (1:20 screening dilution) with radioimmunoprecipitation assay (RIA). Decision limits set at 18% immunomodulation and 100pM respectively. Correlation (Spearman r = 0.897). The RIA values from the seropositive AAG samples from Oxford ranged from 730-3464pM (RI < 100). (B) Correlation of flow cytometric immunomodulation endpoint titer results (decision limit set at positivity starting at 1:20 dilution) and RIA. Correlation (Spearman r = 0.896).