The Multicenter Study of a New Assay for Simultaneous Detection of Multiple Anti-Aminoacyl-tRNA Synthetases in Myositis and Interstitial Pneumonia

<div><p>Objective</p><p>Autoantibodies 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. <b>Methods:</b> We prepared six recombinant ARSs: GST-Jo-1, His-PL-12, His-EJ and GST-KS expressed in <i>Escherichia coli</i>, 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. <b>Results:</b> 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. <b>Conclusion:</b> 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.</p></div

Comparison of the results between the new ELISA system and RNA-IP.

<p>The results detecting the five anti-ARS antibodies (anti-Jo-1, PL-12, EJ, KS, and PL-7) are described (sensitivity: 100%, specificity: 99.8%).</p><p>Numbers in parenthesis are the results detecting all anti-ARS antibodies (including anti-OJ) (sensitivity: 97.1%, specificity: 99.8%).</p

The frequency of each anti-ARS antibody in subsets of IIM.

<p>The frequency of each anti-ARS antibody in subsets of IIM.</p

Antigenic activity of recombinant autoantigens a. Antigenic activity of PL-7 in various conditions.

<p>Left, purified recombinant PL-7 was eluted and diluted in PBS and coated on ELISA plates. Middle and Right, purified recombinant PL-7 was eluted in PBS and diluted in 8M urea and 2 × SDS sample buffer, respectively, and then coated onto ELISA plates. b. Five recombinant ARS antigens (His-PL-12, His-EJ, GST-Jo-1, GST-KS, and His-PL-7) were prepared as soluble polypeptides in PBS and their antigenic activity was tested in an ELISA using sera from five patients each containing corresponding autoantibodies (only GST-KS was tested using sera from three patients). Six healthy controls were used in each ELISA. c. Purified recombinant ARS antigens were electrophoresed on SDS-PAGE and transferred to a PVDF membrane followed by immunoblot analysis. CBB; Coomassie Brilliant Blue staining of gels, M; molecular weight marker, HC; healthy control, Lane 1; His-PL-12, Lane 2; His-EJ, Lane 3; GST-Jo-1, Lane 4; GST-KS and Lane 5; His-PL-7.</p

Confirmation of the efficiency of the ELISA system.

<p>Using the ELISA system, ARS antibodies were measured in 694 serum samples from patients with various CTDs and IIP, and 30 serum samples from healthy controls. The cutoff value (25 U/mL) is indicated by a horizontal dotted line.</p

Comparison of clinical backgrounds between anti-ARS (+) and (−) non-IPF patients.

<p>%VC: % vital capacity, %DLCO: % diffusing capacity of carbon monoxide.</p

The frequency of each anti-ARS antibody in IIM, other CTD and IIP.

<p>The frequency of each anti-ARS antibody in IIM, other CTD and IIP.</p