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

Background and Objectives Autoantibodies against alpha 3-subunit-containing nicotinic acetylcholine receptors (alpha 3-nAChRs), usually measured by radioimmunoprecipitation assay (RIPA), are detected in patients with autoimmune autonomic ganglionopathy (AAG). However, low alpha 3-nAChR antibody levels are frequently detected in other neurologic diseases with questionable significance. Our objective was to develop a method for the selective detection of the potentially pathogenic alpha 3-nAChR antibodies, seemingly present only in patients with AAG. Methods The study involved sera from 55 patients from Greece, suspected for autonomic failure, and 13 patients from Italy diagnosed with autonomic failure, positive for alpha 3-nAChR antibodies by RIPA. In addition, sera from 52 patients with Ca2+ channel or Hu antibodies and from 2,628 controls with various neuroimmune diseases were included. A sensitive live cell-based assay (CBA) with alpha 3-nAChR-transfected cells was developed to detect antibodies against the cell-exposed alpha 3-nAChR domain. Results Twenty-five patients were found alpha 3-nAChR antibody positive by RIPA. Fifteen of 25 patients were also CBA positive. Of interest, all 15 CBA-positive patients had AAG, whereas all 10 CBA-negative patients had other neurologic diseases. RIPA antibody levels of the CBA-negative sera were low, although our CBA could detect dilutions of AAG sera corresponding to equally low RIPA antibody levels. No serum bound to control-transfected cells, and none of the 2,628 controls was alpha 3-CBA positive. Discussion This study showed that in contrast to the established RIPA for alpha 3-nAChR antibodies, which at low levels is of moderate disease specificity, our CBA seems AAG specific, while at least equally sensitive with the RIPA. This study provides Class II evidence that alpha 3-nAChR CBA is a specific assay for AAG. Classification of Evidence This study provides Class II evidence that an alpha 3-nAChR cell-based assay is a more specific assay for AAG than the standard RIPA.