Background/Purpose: The disease expression of dermatomyositis (DM) is highly variable among patients, ranging form those with severe muscle weakness in the absence of internal organ involvement to those with clinically amyopathic DM (CADM) complicating fatal rapidly progressive interstitial lung disease. A series of serum autoantibodies have been identified in DM patients, and are useful in diagnosis as well as disease subsetting. These are antibodies to aminoacyl tRNA synthetase (ARS), including Jo-1, EJ, OJ, PL-7, PL-12, and KS, Mi-2, melanoma differentiation-associated gene 5 (MDA5), transcription intermediary factor-1 (TIF1)-γ, NXP2, SAE, and Ku. These antibodies are detectable by immunorecipitation (IP) assays for RNA and protein components, which require a complicated procedure with cultured human cell lines and a radioisotope. Recently, a novel enzyme-immunoassay (MESACUP anti-ARS test; MBL) has been developed for detection of 5 different anti-ARS specificities together (Jo-1, EJ, PL-7, PL-12, and KS), and a multianalyte line-blot assay that detects Mi-2, Ku, PM-Scl, Jo-1, SRP, PL-7, PL-12, EJ, OJ (Myositis Profile 3 EUROLINE, EUROIMMUN) is commercially available. However, many DM-specific antibodies are still undetectable by these convenient assays. In this study, we have developed a new multianalyte assay for detection of DM-specific antibodies undetectable by current commercial assays, using a principle of IP combined with immunoblots (IB).  

Methods: We enrolled 116 patients with DM, including 52 with CADM, who were diagnosed at our institution between 2000 and 2013. DM-specific antibodies were first identified using IP assays and the MESACUP anti-ARS test. Patients’ sera negative by the MESACUP test were further applied to a multianalyte IP/IB assay, in which native autoantigens were isolated by IP technique, followed by IB probed with a mixture of monoclonal or polyclonal antibodies specific to Mi-2 (240kDa), TIF1γ (155kDa), OJ (150kDa), NXP2 (145kDa), MDA5 (140kDa), SAE (90kDa), and Ku (80kDa). Identification of individual antibodies was based on the molecular sizes of the immunoreactive bands. In some sera, DM-specific specificities were also measured by the EUROLINE.  

Results: DM-specific antibodies were detected in 101 (87%) patients by IP assays. These included MDA5 in 26, Jo-1 in 14, TIF1γ in 14, EJ in 10, PL-7 in 7, PL-12 in 7, OJ in 6, NXP2 in 6, Mi-2 in 4, Ku in 3, SAE in 2, and U1RNP in 2). The MESACUP anti-ARS test was positive in 38 (33%) patients who were completely matched to those positive for Jo-1, EJ, PL-7, or PL-12 by IP assays. When 78 sera negative by the MESACUP test were further subjected to the multianalyte IP/IB assay, results obtained by IP assays and the IP/IB assay were identical in terms of Mi-2, TIF1γ, OJ, NXP2, MDA5, SAE, and Ku (sensitivity and specificity 100%). We also applied 51 sera positive for Mi-2, Ku, Jo-1, PL-7, PL-12, EJ, or OJ to the Euroline, resulting in a false-negative result in all 6 anti-OJ-positive sera and a false-positive result of Ku in 3 sera.  

Conclusion: Multianalyte IP/IB assay is a convenient and reliable method useful for detection of a full panel of DM-specific autoantibodies when it is combined with the commercial MESACUP test.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-new-multianalyte-assay-for-detection-of-dermatomyositis-specific-autoantibodies-undetectable-by-commercially-available-immunoassays/