Development of an assay for the detection of acetylcholine receptor blocking antibodies by Flow Cytometry

Myasthenia Gravis (MG) is a neuromuscular autoimmune disease primarily associated with the presence of antibodies directed toward receptors embedded in the motor end-plate at the neuromuscular junction (NMJ). These antibodies cause blockage, dysfunction, and degradation of acetylcholine receptors (AChR) leading to muscle weakness and fatigability. Three main types of AChR antibodies have been identified and are referred to as binding, modulating, and blocking. Detection of acetylcholine receptor antibodies through the use of a radio-label has become standard procedure in most laboratories. Known drawbacks are associated with radioimmunoassay; cost of radioisotopes, hazards to laboratory professionals, and manufacture and disposal of radioactive materials have prompted investigation into replacement assays. In 2009, scientists described a method of detecting AChR modulating antibodies using flow cytometric techniques. They utilized fluorescently-labeled molecules to aid in the detection of modulated AChR and to confirm the presence of AChR modulating antibodies. We postulate that similar techniques could enable the conversion of the AChR blocking assay to flow cytometry as well. Described here is a high-throughput immunofluorescent flow cytometric assay designed for the detection of AChR blocking antibodies. Three-hundred-twenty-four sera were tested on both the AChR blocking radio-assay and the new immunofluorescent flow cytometric assay. Analysis of the results revealed a 96.9% concordance between the two assay methodologies. Our results indicate that a new immunofluorescent flow cytometric AChR blocking antibody assay is not only feasible, but clinically comparable in both sensitivity (91%) and specificity (99%) when compared to radio-assay.