Myasthenia gravis (MG) is an autoimmune disorder characterized by fatigable skeletal muscle weakness. This can manifest in ocular (ptosis, diplopia), bulbar (dysarthria, hypophonia, dysphagia), axial (limb), face and neck, or respiratory muscle weakness. If unrecognized, it can progress to myasthenic crisis with respiratory failure and death. The symptoms of MG are attributed to autoantibodies against post-synaptic proteins in the neuromuscular junction; these autoantibodies impair signal transmission between nerve and muscle and reduce the endplate potential in muscle fibers. The majority of MG patients (~85%) carry antibodies against acetylcholine receptor (AChR) (Hoch et al., 2001; Lazaridis and Tzartos, 2020). Approximately one third of MG patients seronegative for antibodies to the AChR will have antibodies against muscle-specific kinase (MuSK) (Hoch et al., 2001; Lazaridis and Tzartos, 2020; Gilhus and Verschuuren, 2015). Compared to AChR-MG patients, MuSK-MG patients are more likely to exhibit severe bulbar, facial, and respiratory symptoms early in the course of diseases (Cordts et al., 2017; Rivner et al., 2018). Cholinesterase inhibitors are rarely effective in MuSK-MG patients, and acute treatment requires high doses of corticosteroids; refractory cases may require plasma exchange, and steroid-sparing therapies such as anti-CD20 should be considered early if there is an inadequate response to initial immunotherapy (Rivner et al., 2018; Lavrnic et al., 2005). While AChR-MG may benefit from thymectomy, this has not been established in MuSK-MG (Aljaafari and Ishaque, 2022; Clifford et al., 2019). Given these differences, identification of the causal autoantibody has important implications for the clinical course and treatment of MG.

MuSK is a transmembrane receptor tyrosine kinase expressed on skeletal muscle that plays an important role in signal transduction between the nerve and muscle. When activated by the ligand agrin, the MuSK-LRP4 (low-density lipoprotein receptor-related protein 4) complex signals clustering of AChRs to sites in the neuromuscular junction. In MuSK knockout mice, the neuromuscular junction fails to form properly, leading to respiratory failure and death (DeChiara et al., 1996). In MuSK-MG, MuSK antibodies bind to an Ig-like domain on the MuSK protein and prevent the agrin-MuSK-LRP4 complex from signaling downstream pathways. This disruption reduces AChR density and causes misalignment and deterioration of the neuromuscular junction.

The gold standard in serological MG diagnosis is radioimmunoassay (RIA), both for MuSK and AChR antibodies (Lazaridis and Tzartos, 2020; Benatar, 2006). However, RIA has several limitations that prevent widespread accessibility, including safety and regulatory concerns associated with using radioactive materials. More specifically, the short half-life of the radioactive materials requires complex planning months in advance; insufficient purchasing of reagents will cause delays in patient testing while excessive purchases will result in expiration and waste of radioactive materials.

Alternative methods for detecting MuSK antibodies, such as enzyme-linked immunosorbent assays (ELISA) and fixed cell-based immunofluorescence assays (f-CBA-IFA), have been developed as substitutes for RIA. These alternatives offer several advantages, including the elimination of radioactive materials, longer reagent shelf-life, improved workflow, reduced cost, and less complex laboratory protocols. Recent studies conducted outside the United States have compared ELISA and/or f-CBA-IFA with RIA and demonstrated equivalent specificity and superior sensitivity for detection of MuSK antibodies using cell-based assays (Benatar, 2006; Damato et al., 2022; Kwon et al., 2023; Kim et al., 2021; Han et al., 2019; Villalta et al., 2023).These studies primarily focused on patients previously considered to have double seronegative MG due to negative AChR and MuSK antibody results using RIA. However, studies evaluating the performance of ELISA and f-CBA-IFA in the United States are limited due to previous restrictions on their distribution within the country. Therefore, this study aims to evaluate the diagnostic characteristics of a commercially available f-CBA-IFA and ELISA assay for the qualitative and quantitative determination of MuSK antibodies compared to the RIA methodology.