{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T05:08:28Z","timestamp":1779253708553,"version":"3.51.4"},"reference-count":41,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,8]],"date-time":"2026-05-08T00:00:00Z","timestamp":1778198400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>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\u20132023). 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.<\/jats:p>","DOI":"10.3390\/biom16050698","type":"journal-article","created":{"date-parts":[[2026,5,8]],"date-time":"2026-05-08T20:11:13Z","timestamp":1778271073000},"page":"698","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Clinical Relevance and Methodological Comparison of Anti-MDA5 Antibody Detection: A Five-Year Retrospective and Exploratory Pilot Study"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-3426-5523","authenticated-orcid":false,"given":"S\u00e1ndor","family":"Mogyor\u00f3ssy","sequence":"first","affiliation":[{"name":"Department of Rheumatology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"},{"name":"Doctoral School of Medical Sciences, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2943-2750","authenticated-orcid":false,"given":"G\u00e1bor","family":"Nagy","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1371-7911","authenticated-orcid":false,"given":"Zolt\u00e1n","family":"Griger","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0316-3828","authenticated-orcid":false,"given":"Melinda","family":"Nagy-Vincze","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Monika","family":"Bodoki","sequence":"additional","affiliation":[{"name":"Department of Rheumatology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-5082-3944","authenticated-orcid":false,"given":"D\u00f3ra","family":"Csige","sequence":"additional","affiliation":[{"name":"Department of Rheumatology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"T\u00fcnde","family":"Tarr","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"\u00c9va","family":"Z\u00f6ld","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gy\u00f6rgy","family":"Pfliegler","sequence":"additional","affiliation":[{"name":"Centre of Rare Diseases, Department of Internal Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bogl\u00e1rka Csilla","family":"Br\u00fag\u00f3s","sequence":"additional","affiliation":[{"name":"Centre of Rare Diseases, Department of Internal Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hui","family":"Lu","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, University of Bath, Bath BA2 7AY, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sarah","family":"Tansley","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, University of Bath, Bath BA2 7AY, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"P\u00e9ter","family":"Antal-Szalm\u00e1s","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-3676-9911","authenticated-orcid":false,"given":"Andrea","family":"Domj\u00e1n","sequence":"additional","affiliation":[{"name":"Department of Rheumatology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Szilvia","family":"Szamosi","sequence":"additional","affiliation":[{"name":"Department of Rheumatology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0958-9531","authenticated-orcid":false,"given":"Gabriella","family":"Sz\u0171cs","sequence":"additional","affiliation":[{"name":"Department of Rheumatology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zolt\u00e1n","family":"Szekanecz","sequence":"additional","affiliation":[{"name":"Department of Rheumatology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"\u00c1gnes","family":"Horv\u00e1th","sequence":"additional","affiliation":[{"name":"Department of Rheumatology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Levente","family":"Bodoki","sequence":"additional","affiliation":[{"name":"Department of Rheumatology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary"},{"name":"Doctoral School of Medical Sciences, University of Debrecen, 4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Yoshida, A., Nagata, S., Okazaki, Y., Hanaoka, H., Gono, T., and Kuwana, M. 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Highlights"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/5\/698\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T04:36:30Z","timestamp":1779251790000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/5\/698"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,8]]},"references-count":41,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["biom16050698"],"URL":"https:\/\/doi.org\/10.3390\/biom16050698","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,8]]}}}