A cell-based assay's construction for the detection of acetylcholine receptor

Abstract

Myasthenia Gravis (MG) is an autoimmune disease resulting from the action of autoantibodies against nicotinic acetylcholine receptors (nAChRs), interfering with the proper communication between the neurotransmitter acetylcholine (ACh) released by the axon and its nAChR receptor, expressed by the muscle fiber. The detection of anti-nAChR autoantibodies has 100% specificity for the diagnosis of MG, however the traditional methodologies for anti-nAChR detection needs improvement, as they either have very low sensitivity, such as the ELISA (Enzyme Linked Immunoassay) that uses recombinant antigens, or are based on the use of radioisotopes, such as the RIPA (RadioImmuno Precipitation Assay), with execution restricted to some laboratories resulting in high costs.Our objective is to develop a cell-based assay (CBA) for the detection of anti-nAChRs antibodies in samples from patients with MG and to evaluate its performance in relation to traditional methodologies.The research is divided in two stages: 1- construction of vector plasmids with the genes for the five subunits of nAChR, followed by transfection to generate a HEK293T cell lineage that expresses the nAChR receptor on its membrane in a stable manner. These cells will be used as a substrate for an indirect immunoflorescence reaction (IIF) using the sample of the patient with MG as the primary probe; 2- For validation of the assay, 100 samples will be collected from patients diagnosed with MG and subjected to analysis for the presence of anti-AChR in our CBA assay as well as by RIPA and ELISA.We expect to develop a test for detection of anti-AChR of simplified execution, similar to an IIF reaction, that presents superior sensitivity then the ELISA and at least similar to RIPA. (AU)