The multicenter study of a new assay for simultaneous detection of multiple anti-aminoacyl-tRNA synthetases in myositis and interstitial pneumonia

Abstract

Objective: 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.

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.

Figure 1. Antigenic activity of recombinant autoantigens a. Antigenic activity of PL-7 in various conditions.

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.

Figure 2. Confirmation of the efficiency of the ELISA system.

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.