Detection of autoantibodies against the acetylcholine receptor, evaluation of commercially available methodologies: fixed Cell-Based Assay, Radioimmunoprecipitation Assay and Enzyme-Linked Immunosorbent Assay

Introduction Myasthenia Gravis (MG) is an autoimmune disease resulting from the action of pathogenic autoantibodies (AAbs) directed against nicotinic acetylcholine receptors (AChR), which interfere with communication between the neurotransmitter acetylcholine and its receptor on the muscle fiber. The detection of anti-AChR using Radio Immuno Precipitation Assay (RIPA) has 100% specificity for the diagnosis of MG, however RIPA has high execution and interpretation complexity and requires radioactive materials, which restrict their use to specialized laboratories. Objective We compared the performance of the gold standard RIPA with different non-RIPA anti-AChR immunoassays, including a cell-based assay (CBA) and two solid-phase ELISA kits. Results 145 samples were included with medical indication for anti-AChR testing. By the RIPA method, 63 were negative (RIPA-Neg <0.02 nmol/L), 17 were classified as B orderline (≥0.02 – 1 nmol/L), and 65 were positive (RIPA-Pos >1 nmol/L). The competitive ELISA yielded a poor performance with low Kappa agreement with RIPA (0.210). The indirect ELISA yielded a substantial Kappa agreement (Kappa=0.652), with ∼70% sensitivity and ∼96% specificity, compared to RIPA. In a semiquantitative analysis, there was a good Spearman correlation between the indirect ELISA and RIPA levels (r=0.845). The best performance was observed with the CBA that uses fixed cells expressing clustered AChR as antigenic substrate. There was an almost perfect agreement with RIPA (Kappa = 0.969), with ∼97% sensitivity and 100% specificity. However, in the Borderline group, only 5 (∼30%) were positive using the CBA method, suggesting a slightly lower sensitivity for the CBA. Conclusion For detection of anti-AChR reactivity, the indirect immunofluorescence assay yielded a very good analytical performance taking RIPA as the reference method, with potential to replace the RIPA in the clinical laboratory. ELISA could be an option to estimate anti-AChR AAb levels after confirming positivity by the CBA. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was mainly supported by Sao Paulo Government agency FAPESP (Sao Paulo State Research Foundation) grant numbers #2017/20745-1 and #2021/04588-9, granted to G.D.K. and L.D.S. Also, G.D.K. is supported by the Vicerrectoria de Investigacion y Desarrollo Tecnologico (VRIDT) from the Universidad Catolica del Norte (UCN). Additionally, L.E.C.A. is supported by the Brazilian research agency National Council for Research (CNPq), grant #PQ-1D 310334/2019-5. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of Fleury Group gave ethical approval for this work (Plataforma Brasil CAAE: 57480622.3.0000.5474). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.