Detection of acetylcholine receptor modulating antibodies by Flow Cytometry

Myasthenia gravis is an autoimmune disease characterized by antibodies against acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ) of skeletal muscle. These antibodies interfere with the transmission of nerve impulses resulting in weakness and paralysis. Due to the variability in symptoms and heterogeneity of autoantibody production in patients with myasthenia gravis, it is essential to make available the best clinical laboratory tests to aid the clinician in diagnosis. One means by which AChR antibodies interfere with nerve impulse transmission is through the effect of antigenic modulation, a process in which antibody-bound AChRs on the postsynaptic muscle cell membrane are internalized and destroyed. The current laboratory assay for the detection of AChR modulating antibodies involves measuring the reduction of expression of radiolabeled AChRs on a human rhabdomyosarcoma (RD) cell line in response to exposure to patient serum. The goal of this study was to determine the feasibility of detection of AChR modulating antibodies by a new flow-cytometric method rather than the current radioimmunoassay. Two cell lines were investigated: the RD cell line which expresses fetal AChRs and the DB40 cell line which has been transfected with genes for the expression of fetal and adult acetylcholine receptors. Samples tested included sera from 120 self-proclaimed healthy individuals and 100 samples submitted for clinical testing, iv 50 of which were AChR antibody positive and 50 of which were AChR antibody negative. Results of the flow-cytometric AChR modulating antibody testing on the RD cell line correlated best with results for currently available assays and demonstrated better sensitivity and specificity than the current radioimmunoassay. Results of AChR modulating antibody testing on the DB40 cell line showed slightly decreased sensitivity and specificity, potentially resulting from defects in receptor metabolism due to gene transfection. Detection of AChR modulating antibodies by flow cytometry is feasible and uses an assay format that is more sensitive, specific, and robust with less cost and environmental burden.