The presence of anti-ENA antibodies was screened by a commercially available line immunoassay (ANA 3 Profile EUROLINE, Euroimmun, Lübeck, Germany). The assay was performed according to the manufacturer's instructions. The sera were diluted 1 : 100 in sample buffer. After the first incubation with diluted serum, a second incubation was performed with goat anti-human IgG linked to alkaline-phosphatase. Finally, a third incubation took place with bromochloroindolyl phosphate and nitro blue tetrazolium chloride (BCIP/NBT) as substrate to detect anti-ENA antibodies. Although the ANA 3 Profile EUROLINE kit enables detection of 15 different antigens, only eight were evaluated in the current study: Ro52, SS-A/Ro60, SS-B/La, nRNP/Sm, Sm, Scl-70 (topoisomerase 1), Jo-1, and CENP-B. Reading of the results was automated and the colour intensities of the reactions were evaluated by the EUROLineScan program (Euroimmun) to enable semiquantitative determination, that is, equivocal, 1+, 2+, and 3+. Results were considered positive for Sm, Scl-70, and Jo-1 if the intensity was at least equivocal, while for SS-A/Ro60, SS-B/La, nRNP/Sm, and CENP-B an intensity of at least 1+ was required. Finally, the cutoff for Ro52 was 2+.
Euroline ana profile 3
Manufactured by EUROIMMUN
Sourced in Germany
The Euroline ANA Profile 3 is a laboratory diagnostic test kit designed to detect and identify autoantibodies associated with various autoimmune disorders. The test utilizes a line assay format to provide qualitative and semi-quantitative results.
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4 protocols using euroline ana profile 3
1
Anti-ENA Antibodies Detection Protocol
2
Autoantibody Profiling in Primary Sjögren's Syndrome
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The immunological data of the pSS patients, including the levels of serum autoantibodies, rheumatoid factor (RF), immunoglobulins, and complement component 3 (C3), were extracted. Serum autoantibodies were determined using an immunoblot assay kit (EUROLINE: ANA Profile 3, EUROIMMUN, Lübeck, Germany) that could quantitatively detect 15 autoantibodies including antibodies for nRNP/Sm, Sm, SSA (Ro60), Ro52, SSB, Scl-70, PM-Scl, Jo-1, centromere protein B (CENP-B), proliferating cell nuclear antigen (PCNA), nucleosomes, histones, ribosomal P-proteins, mitochondrion, and intractable cellular antigen dsDNA. The antibody intensity levels were divided into four grades according to the grayscale values of the immunoblot assay, determined using the EUROLineScan software. Specifically, grayscale values from 0 to 10 were defined as negative. Grayscale values greater than 10 were defined as positive: grayscale values from 11 to 50 were scored as 1+ (grade 1), from 51 to 90 as 2+ (grade 2), and those greater than 90 as 3+ (grade 3). RF level was determined using an immunoturbidimetric latex assay (Jiuqiang Biological Co., Ltd. China). Immunoglobulins (Ig)G, M, and A, and C3 levels were determined by immunoscatter turbidimetry (Zhongsheng Beikong Biological Co., Ltd., China).
3
Autoantibody Screening in Anti-DFS70 Positive Sera
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To examine potential association with other autoantibodies, sera positive for anti-DFS70 by ELISA and LIA were further screened for fifteen autoantibodies (nRNP/Sm, Sm, SSA/Ro60, Ro52/TRIM21, SSB/La, Scl-70, PM-Scl, Jo-1, CENP-B, PCNA, dsDNA, nucleosomes, histones, ribosomal P protein (Rib-P), AMA-M2) using the Euroline ANA Profile 3 (Euroimmun) according to the manufacturer’s instructions.
4
Screening for Autoantibody Profiles
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To explore the potential association with other autoantibodies, partial sera positive for anti-RR by LIA were further screened for fifteen autoantibodies (nRNP/Sm, Sm, SSA/Ro60, Ro52/TRIM21, SSB/La, Scl-70, PM-Scl, Jo-1, CENP-B, PCNA, dsDNA, nucleosomes, histones, ribosomal P protein (Rib-P), AMA-M2) using the Euroline ANA Profile 3 (Euroimmun, Germany) according to the manufacturer’s instructions (27 (link)).
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