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Multicenter Study
. 2016 Apr 26;11(4):e0154285.
doi: 10.1371/journal.pone.0154285. eCollection 2016.

Clinical Utility of an Enzyme-Linked Immunosorbent Assay for Detecting Anti-Melanoma Differentiation-Associated Gene 5 Autoantibodies

Shinji Sato  1 , Akihiro Murakami  2 , Akiko Kuwajima  2 , Kazuhiko Takehara  3 , Tsuneyo Mimori  4 , Atsushi Kawakami  5 , Michiaki Mishima  6 , Takafumi Suda  7 , Mariko Seishima  8 , Manabu Fujimoto  9 , Masataka Kuwana  10
Affiliations
Multicenter Study

Clinical Utility of an Enzyme-Linked Immunosorbent Assay for Detecting Anti-Melanoma Differentiation-Associated Gene 5 Autoantibodies

Shinji Sato et al. PLoS One. .

Abstract

Objective: Autoantibodies to melanoma differentiation-associated gene 5 (MDA5) are specifically expressed in patients with dermatomyositis (DM) and are associated with a subset of DM patients with rapidly progressive interstitial lung disease (RP-ILD). Here, we examined the clinical utility of a newly developed enzyme-linked immunosorbent assay (ELISA) system for detecting these antibodies.

Methods: Here we developed an improved ELISA for detecting anti-MDA5 antibodies. We then performed a multicenter clinical study involving 8 medical centers and enrolled 242 adult patients with polymyositis (PM)/DM, 190 with non-PM/DM connective tissue disease (CTD), 154 with idiopathic interstitial pneumonia (IIP), and 123 healthy controls. Anti-MDA5 antibodies in the patients' serum samples were quantified using our newly developed ELISA, and the results were compared to those obtained using the gold-standard immunoprecipitation (IP) assay. In addition, correlations between the ELISA-quantified anti-MDA5 antibodies and clinical characteristics were evaluated.

Results: In patients with PM/DM, the anti-MDA5 antibody measurements obtained from the ELISA and IP assay were highly concordant; the ELISA exhibited an analytical sensitivity of 98.2%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.5% (compared to the IP assay). Anti-MDA5 antibodies were detected in 22.7% of the DM patients, but not in any of the patients with PM, non-PM/DM CTD, or IIP. Clinically amyopathic DM, RP-ILD, arthritis, and fever were more prevalent in DM patients who were anti-MDA5 antibody-positive than in those who were antibody-negative (P ≤ 0.0002 for all comparisons). In addition, anti-MDA5 antibody-positive patients with RP-ILD exhibited higher antibody levels than those without RP-ILD (P = 0.006).

Conclusion: Our newly developed ELISA can detect anti-MDA5 antibodies as efficiently as the gold standard IP assay and has the potential to facilitate the routine clinical measurement of anti-MDA5 antibodies in patients who suspected to have DM.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: Masataka Kuwana, the corresponding author, currently serves as an Academic Editor of PLOS ONE. This does not alter the authors' adherence to all of the PLOS ONE’s policies on sharing data and materials. S. Sato and M. Kuwana hold a patent on the anti-MDA5 antibody measurement system. A. Murakami and A. Kuwajima are employees of Medical and Biological Laboratories. This study was conducted as a collaboration between the authors’ institutes and Medical and Biological Laboratories. The remaining authors state that they have no other conflict of interest.

Figures

Fig 1
Fig 1. Purity and antigenicity of recombinant GST-MDA5 protein.
A. Purified recombinant GST-MDA5 fusion protein (2 μg) was subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis, followed by 0.05% Coomassie blue staining. The GST-MDA5 fusion protein was detected as a single band at ~166 kDa (arrow). B. Immunoblot analysis of the recombinant fusion protein was performed by probing with anti-MDA5 antibody-positive DM sera (lanes 1–3), anti-MDA5 antibody-negative DM sera (lanes 4–6), an anti-GST polyclonal antibody (lane 7), and an anti-MDA5 monoclonal antibody (lane 8).
Fig 2
Fig 2. ROC curve analysis to determine the optimal cut-off value for ELISA-quantified anti-MDA5 antibodies.
A. The ROC curve showed high concordance between the ELISA and IP assay (area under the curve > 0.99, P < 0.0001). B. The sensitivity and specificity of the anti-MDA5 antibody ELISA for various cutoff levels. A cutoff of 32 U/mL (arrow) provided a sensitivity of 98.2% and specificity of 100%.
Fig 3
Fig 3
Anti-MDA5 (A) and anti-ARS (B) antibody levels in patients with PM/DM, non-PM/DM CTD, IIP, and healthy controls. Anti-MDA5 and anti-ARS antibodies were measured by ELISA in the sera from 242 patients with PM/DM (70 PM, 104 classic DM, and 68 CADM), 190 patients with non-PM/DM CTD, 154 patients with IIP, and 123 healthy volunteers. Cutoff levels of anti-MDA5 and anti-ARS antibodies are shown as broken lines (32 and 25 U/mL, respectively).
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References

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