Fluorescence-detection size-exclusion chromatography specifically detects autoantibodies targeting the ganglionic acetylcholine receptor in patients with autoimmune autonomic ganglionopathy

Abstract

Autoimmune autonomic ganglionopathy (AAG) is a rare disease wherein autoantibodies target the ganglionic acetylcholine receptor (gAChR). Current diagnosis in the United States depends upon clinical symptoms and positive autoantibody detection using a radioimmunoprecipitation assay (RIA). Here we offer a proof-of-principle study on an alternative method, fluorescence-detection size-exclusion-chromatography (FSEC). We show FSEC can detect autoantibodies against gAChR from patient sera but not healthy controls or samples from other autoimmune diseases. We compare FSEC to RIA and find good correlation. We discuss potential advantages of using FSEC as an alternative or as a first-step diagnostic prior to pursuing existing methodologies.

Keywords: Acetylcholine receptor; Autonomic; Detection; FSEC; Membrane protein; Neuronal.

Figure 1:. Overview of FSEC method to detect autoantibodies in patients with autoimmune autonomic ganglionopathy

A, Workflow of HEK-cell production of gAChR-GFP pentameric receptors. B, FSEC of α3β4-GFP (pentamer peak measurement taken at black dashed line). Orange trace is pooled human serum, black trace is untransfected lysate. Green traces are serial 3-fold dilutions of a previously characterized monoclonal antibody against gAChR13. The antibody was diluted in human serum to allow for nonspecific reduction of pentameric signal, from 0.06 µg/ml (darkest green) to 0.0003 µg/ml (lightest green). The horizontal lines underneath the graph are to illustrate the various peaks one may encounter during SEC; line 1 represents the void/aggregate peaks, line 2 is the receptor as predetermined by purified protein, line 3 are autofluorescent small molecules found in serum and cell lysate.

Figure 2:. AAG patients selectively reduce gAChR.

A, Percent reduction of the gAChR pentamer signal as described in Methods is plotted for 12 healthy controls (HC), 15 autoimmune autonomic ganglionopathy patients with RIA >200 pM (AAG RIA+), 5 AAG patients with seronegative AAG or AAG treated with immunotherapy (AAG RIA−), 18 patients with postural orthostatic tachychardia syndrome (POTS), 20 myasthenia gravis patients (MG), 4 autoimmune encephalitis patients (AE), and 5 patients with other neurological diseases that do not test positive for gAChR antibodies. B, Representative FSEC run for an AAG patient (red traces) vs. healthy control (black traces) with the α7 nAChR. Inset shows full AAG+ RIA+ patient data.

Figure 3:. The FSEC assay is specific for symptomatic AAG.

A, FSEC data from 2-fold serial dilutions for the 15 AAG patients that meet the decision limit cutoff. B, FSEC Data from one patient pre- and post-treatment C, Correlation between FSEC percent reduction and radioimmunoprecipitation assay. Vertical dotted line indicates accepted cutoff for RIA of 50, 100 and 200 pM; horizontal dotted line is decision limit cutoff for FSEC of 23.5%. Patients with negative RIA were tested for gAChR because of suspicion of AAG; these include the patients of the POTS and treated/seronegative cohorts.

Figure 4:. Receiver operator characteristic curve comparing AAG patients to all others at different decision limit cutoffs.

Y-axis is the true positive fraction; X-axis is false positive fraction. Ratios of TPF/FPF were determined at 0–100% reduction cutoffs in 10% increments.