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

Abstract

AbstractIntroductionMyasthenia 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.ObjectiveWe 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.Results145 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 Borderline(≥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 theBorderlinegroup, only 5 (∼30%) were positive using the CBA method, suggesting a slightly lower sensitivity for the CBA.ConclusionFor 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.