Eurolinescan

The Euroline myositis line-blot assay of Euroimmun (Lübeck, Germany) was performed according to the instructions of the manufacturer. This blot allows for detection of multiple MSA, namely antibodies against SRP, EJ, OJ, Mi-2α, Mi-2β, TIF1-γ, MDA5, NXP2, SAE1, PL-12, PL-7 and Jo-1, and MAA, specifically antibodies against Ku, PM/Scl-75 and PM/Scl-100. Anti-Ro52 was excluded from the analysis because anti-Ro52 is not specific for myositis or connective tissue diseases [12 (link)]. HGMCoAR antibodies and cN1A antibodies have not been systematically tested in this cohort of patients, since they are not part of the above-mentioned assay.
All immunoblot strips were analyzed with the EUROLineScan (Euroimmun) according to the manufacturers recommendations, and scored negative, weakly positive (+) and positive (++ or +++). This corresponds to intensity levels 0–10, 11–25 and ​> ​25, respectively. The strong positive intensity level (>50) was not reported by all centers, therefore all intensity levels >25 were included in the positive category.

Samples were collected at the time of study enrollment, and immunoprecipitation assay tests were performed simultaneously. The presence of myositis antibodies was assessed using the immunoblot assay Euroline: Autoimmune Inflammatory Myopathies 16Ag (Euroimmun, Bussy-Saint-Martin, France). This assay utilizes membrane strips with antigens to detect the presence of myositis-related antibodies in patient sera. The immunoblot detects antibodies to the following antigens: Ku, PM/Scl100, PM/Scl75, Jo-1, SRP, PL-7, PL-12, EJ, OJ, Ro-52, Mi-2 α/β, TIF1γ, MDA5, NXP2, and SAE1. Because both anti-Mi-2α and Mi-2β target two closely related isoforms of the same protein, they were considered together as anti-Mi-2.8 (link) Similarly, anti-PM/Scl100 and anti-PM/Scl75 were both considered as anti-PM/Scl. All immunoblot strips were analyzed with Euroline Scan (Euroimmun), which provides semi-quantitative results based on signal intensities measured for each Ab. We chose to exclude borderline positivity from our study. The analysis of the results was performed semi-quantitatively based on the signal intensity of each antibody, following the manufacturer's recommendations. Anti-nuclear antibodies (ANA) were assessed by indirect immunofluorescence on HEp-2 cells. The assay was performed according to the manufacturer's recommendations using a screening dilution of 1:80.