Abstract
ObjectiveAutoantibodies to aminoacyl-tRNA synthetases (ARSs) are useful in the diagnosis of idiopathic inflammatory myopathy (IIM) with interstitial pneumonia (IP). We developed an enzyme-linked immunosorbent assay (ELISA) system using a mixture of recombinant ARS antigens and tested its utility in a multicenter study. Methods: We prepared six recombinant ARSs: GST-Jo-1, His-PL-12, His-EJ and GST-KS expressed in Escherichia coli, and His-PL-7 and His-OJ expressed in Hi-5 cells. After confirming their antigenic activity, with the exception of His-OJ, we developed our ELISA system in which the five recombinant ARSs (without His-OJ) were mixed. Efficiency was confirmed using the sera from 526 Japanese patients with connective tissue disease (CTD) (IIM n = 250, systemic lupus erythematosus n = 91, systemic sclerosis n = 70, rheumatoid arthritis n = 75, Sjögren’s syndrome n = 27 and other diseases n = 13), 168 with idiopathic interstitial pneumonia (IIP) and 30 healthy controls collected from eight institutes. IIPs were classified into two groups; idiopathic pulmonary fibrosis (IPF) (n = 38) and non-IPF (n = 130). Results were compared with those of RNA immunoprecipitation. Results: Sensitivity and specificity of the ELISA were 97.1% and 99.8%, respectively when compared with the RNA immunoprecipitation assay. Anti-ARS antibodies were detected in 30.8% of IIM, 2.5% of non-myositis CTD, and 10.7% of IIP (5.3% of IPF and 12.3% of non-IPF). Anti-ARS-positive non-IPF patients were younger and more frequently treated with glucocorticoids and/or immunosuppressants than anti-ARS-negative patients. Conclusion: A newly established ELISA detected anti-ARS antibodies as efficiently as RNA immunoprecipitation. This system will enable easier and wider use in the detection of anti-ARS antibodies in patients with IIM and IIP.
Highlights
A number of autoantibodies can be detected in sera from patients with idiopathic inflammatory myopathy (IIM), some of which are specific to IIM
Because we hypothesized that poor antigenic activity of recombinant PL-7 and OJ was due to a lack of posttranslational modification or proper structural folding, we prepared both fusion proteins expressed in eukaryotic Hi-5 cells using the baculovirus system
We tested a variety of antigen mixtures to estimate the most appropriate ratio and concentration to use, and we found that anti-aminoacyl-tRNA synthetases (ARSs)-positive sera showed reactivity with all five different ARSs with the highest sensitivity and specificity occurring at antigen concentrations of 0.63, 1.25, 1.25, 0.63, and 2.5 mg/mL (6.25 mg/mL in total) for histidyl, threonyl, alanyl, glycyl, and asparaginyl-tRNA synthetases, respectively
Summary
A number of autoantibodies can be detected in sera from patients with idiopathic inflammatory myopathy (IIM), some of which are specific to IIM (known as myositis-specific autoantibodies: MSAs). Detection of these autoantibodies is closely associated with IIM clinical manifestations [1,2]. Patients with anti-ARSs show a spectrum of common clinical manifestations known as anti-synthetase syndrome (ASS), including myositis, interstitial pneumonia (IP), nonerosive arthritis, fever, Raynaud’s phenomenon, and mechanic’s hands. Anti-ARS antibodies are useful in diagnosing IIM and in predicting late-onset myopathy in IPproceeding patients and the clinical course of IP in myositis
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