A multianalyte assay for the detection of dermatomyositis-related autoantibodies based on immunoprecipitation combined with immunoblotting.

Abstract

OBJECTIVE:

To develop a multianalyte assay for the detection of dermatomyositis (DM)-related autoantibodies using immunoprecipitation (IP) combined with immunoblotting (IB).

METHODS:

Sera from 116 DM patients were subjected to RNA and protein immunoprecipitation assays as well as commercial enzyme-linked immunosorbent assays (ELISAs) for anti-aminoacyl transfer RNA synthetase, anti-melanoma differentiation antigen 5 (MDA5), anti-Mi-2, anti-transcriptional intermediary factor-1γ (TIF-1γ), and anti-U1 ribonucleoprotein antibodies. The IP/IB assay was developed by immunoprecipitation of autoantigens from HeLa cell extracts using patient sera, followed by immunoblotting with an antibody against Mi-2, TIF-1γ, OJ, nuclear matrix protein (NXP)-2, MDA5, PM/Scl, small ubiquitin-like modifier activating enzyme (SAE), or Ku. A multianalyte assay was designed by mixing primary antibodies in the IP/IB assay.

RESULTS:

IP assays identified any DM-related autoantibodies in 100 patients (86%), of which 82% were covered by commercial ELISAs, with a false-positive result in two sera and a false-negative result in one serum. The results obtained from the multianalyte IP/IB assay and 'gold-standard' IP assays were concordant in terms of the presence or absence of anti-MDA5, anti-TIF-1γ, anti-OJ, anti-NXP-2, anti-PM/Scl, anti-SAE, anti-Mi-2, and anti-Ku antibodies.

CONCLUSION:

This multianalyte IP/IB assay combined with commercial ELISAs is an alternative to 'gold-standard' IP assays for the detection of DM-related autoantibodies.

• Authors: Masataka Kuwana, Yuka Okazaki
• Journal: Modern rheumatology (Mod Rheumatol) Vol. 33 Issue 3 Pg. 543-548 (Apr 13 2023) ISSN: 1439-7609 [Electronic] England
• PMID: 35662349 (Publication Type: Journal Article)
• Copyright: © Japan College of Rheumatology 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected].

Chemical References

• Autoantibodies
• Biomarkers
• Antibodies, Antinuclear

Topics

• Humans
• Dermatomyositis
• HeLa Cells
• Autoantibodies
• Immunoprecipitation
• Immunoblotting
• Biomarkers
• Antibodies, Antinuclear