Development of an assay for modulating anti-acetylcholine autoantibodies using a human rhabdomyosarcoma cell line

Myasthenia gravis (MG) is an autoimmune disease that affects the neuromuscular transmission at the synaptic junction. A polyclonal autoantibody response is elicited against the acetylcholine receptors (AChR) of skeletal muscle that either blocks or eliminates the AChR. MG is estimated to affect between 50 to 125 million people worldwide. The three types of autoantibodies directed against the AChR that are detectable in patients with MG are binding, blocking and modulating. Each autoantibody is characterized by its functional interaction with the AChR. Modulating autoantibodies correlate best with the severity of the disease, but measurement of these antibodies is technically difficult and generally requires the use of primary human muscle cell cultures. An assay to determine the extent of AChR modulation using a rhabdomyosarcoma (RD) cell line was developed to replace the use of live primary human muscle cell cultures. The cell surface AChR expression in RD cells was stimulated by decreasing the concentration of FetalClone 111 serum from 10% to 0.5% in Eagles Essential Medium (EMEM) thereby increasing the sensitivity of the assay. The extent of modulation was determined as the percent of AChR internalized in the presence or absence of modulating autoantibodies. Normal human serum resulted in less than 6 % modulation (N=42). When 105 clinical samples were assayed in our laboratory and at Nichols Institute, a correlation coefficient of 0.816 was obtained. Intraassay and interassay precision studies were also performed resulting in less than 20% variation. Due to several important factors - the utilization of RD cells, an increased number of AChR on the cell surface of the RD cells that resulted from a reduced concentration of FetalClone III, elimination of all bovine serum during the assay and the high specific activity of the U5I-butx - small amounts of modulating autoantibodies were detectable. These improvements allowed for the development of a modulating assay that can be used in clinical laboratories for the diagnosis and management of MG patients.