B-087 Ganglionic Nicotinic Acetylcholine Receptor Binding Antibody Flow Cytometry Assay

Background To develop and validate a sensitive live cell-binding assay using flow cytometry to detect a3ß4-ganglionic nicotinic acetylcholine receptor (gnAChR) antibodies for diagnosis of autoimmune autonomic ganglionopathy (AAG, a subset of autoimmune autonomic neuropathies). The current radioimmunoprecipitation assay (RIPA) method has high false positive rate and utilizes radioactivity. Methods HEK293 cells were transiently transfected to express human a3ß4-gnAChR subunits, along with NACHO and RIC3 chaperons, and GFP for detection. Transfected cells were treated with 1mM nicotine liquid for 18-22 hours prior to lifting cells for flow cytometry. The IBI (IgG Binding Index) was then calculated as the median fluorescence intensity (MFI) of AF647 of the a3ß4-gnAChR subunits expressing cells divided by the MFI of the AF647 HEK293 expressing cells. Humanized AChR-specific monoclonal antibody (mAb03) was used as a single-point calibrator. The cut-off index (COI) ratio is calculated by dividing the IBI obtained from a sample by the established IBI cut-off value of the mAB03 calibrator set by the assay. A ratio >1 indicates a positive result, while a ratio below 1 is negative. The study tested sera from 83 Mayo Clinic patients suspected of having autoimmune autonomic ganglionopathy (AAG), with a total of 100 samples collected over several years (40 from patients with clinically confirmed AAG, and 60 from patients without AAG), all previously positive for a3-gnAChR antibodies by RIPA. Additionally, 130 control samples were included: 90 from healthy individuals, 22 with hypergammaglobulinemia, and 18 with systemic lupus erythematosus. Results Median RIPA a3ß4-gnAChR value among 40 AAG patient samples was 2.58 nmol/L (range, 0.06-29.1 nmol/L) and among 60 non-AAG patients was 0.14 nmol/L (range, 0.01-4.43 nmol/L). The mAB03 calibrator median IBI value across assays was 2.1 (range, 1.6-2.7). The COI ratio was calculated using the calibrated IBI values. Thirty-eight of 40 AAG patient samples were considered positive, resulting in median IBI of 3.10 (range, 1.29-6.71); sensitivity 95%. None of non-AAG patients and controls were considered positive; median IBI of 0.38 (range, 0.14-0.51); specificity 100%. The two negative AAG patient samples (IBI, 0.43 and 0.43) were from patients in remission (treated with immune suppressants), and had RIPA titers of 0.06 nmol/L and 0.74 nmol/L. Conclusion The a3ß4-gnAChR-IgG flow cytometry assay has high sensitivity and specificity for AAG-related a3-AChR antibodies. It was able to distinguish AAG patients from non-AAG patients and controls effectively. The flow cytometry assay’s cut-off index ratio effectively identifies AAG patients while remaining negative for non-AAG and control samples.