Clinical Relevance and Methodological Comparison of Anti-MDA5 Antibody Detection: A Five-Year Retrospective and Exploratory Pilot Study

Anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibodies are critical biomarkers in myositis, associated with distinct clinical features and prognosis. This study aimed to evaluate the proportion of anti-MDA5 positivity and compare the diagnostic performance of local immunoblotting (IB) with gold-standard immunoprecipitation (IP). We performed a retrospective analysis of 3272 physician-requested anti-MDA5 IB determinations over a five-year period (2019–2023). A subsequent exploratory pilot study of ten Hungarian patients with myositis was conducted to compare IB results with radiolabeled protein IP. Confirmatory in-house enzyme-linked immunosorbent assay (ELISA) was used to distinguish between 140 kDa bands (anti-MDA5 vs. anti-NXP2). Indirect immunofluorescence (IIF) on HEp-2 cells was also evaluated. In the retrospective cohort, 3.7% (n = 121) of samples were non-negative. Among 64 borderline patients, only one (1.6%) had a definitive diagnosis of dermatomyositis (DM). Conversely, the proportion of confirmed myositis cases was notably higher among patients with strong positive IB results. In our exploratory cross-sectional pilot study, complete concordance between the two assays was observed for negative and strong positive results. Discrepancies were noted in borderline and weak positivity ranges, where anti-MDA5 was not detected by IP; instead, alternative autoantibodies were identified. The three IP-confirmed MDA5 positive samples were all validated by ELISA. The characteristic IIF cytoplasmic staining was identifiable in 2 out of 10 cases (20%). In our cohort, borderline IB cases were frequently potential false positives, highlighting the need for careful clinical evaluation. Borderline and weak results require clinical correlation or confirmatory testing to avoid misdiagnosis.