The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.
Antibodies/*genetics ; Antibodies/immunology ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Histidine-tRNA Ligase/biosynthesis ; Histidine-tRNA Ligase/*genetics ; Histidine-tRNA Ligase/immunology ; Open Reading Frames/genetics ; Prokaryotic Cells/metabolism

BACKGROUND: The detection of specific autoantibodies in the sera of patients with systemic rheumatic diseases plays a key role in the differential diagnosis. Enzyme-linked immunosorbent assays (ELISA) is known as sensitive and semiquantitative method to detect autoantibodies and ELISA kits using a recombinant fusion protein as antigen have been developed. So, various commercial ELISA have recently become available in a diagnostic laboratory. We investigated the clinical value of antinuclear autoantibodies using commercial ELISA kits. METHODS: The serum of 90 patients were tested for autoantibodies to SSA/Ro, SSB/La, nRNP/Sm and Sm antigens by ELISA using four commercial kits, EL-ANA(TM) (TheraTest, IL, USA), DIASTAT(TM)(SHIELD, DUNDEE, UK), QUANTALit(TM) (INOVA, CA, USA), Varelisa(TM) (elias, WI, USA). We evaluated the clinical usefulness of panel test of Varelisa(TM) in the diagnosis of systemic rheumatic diseases. RESULTS: The concordance rates of four ELISA kits for autoantibodies to SSA/Ro, SSB/La, RNP/Sm and Sm antigens were 83.6%, 74.5%, 87.5% and 80.0%, respectively. Using panel test of Varelisa(TM), positive rates of autoantibodies to Ul-snRNP, nRNP/Sm, Sm, SSA/Ro, SSB/La, Scl-70, CENP and Jo-1 antigens in SLE were 30.0, 40.0, 33.3, 46.7, 20.0, 20.0, 10.0, and 0%, respectively. Of 30 patients with SLE, 16 (53.3%) were positive for 2 or more antibodies. CONCLUSIONS: EL-ANA(TM), QUANTALite(TM) and Varelisa(TM) show more positive rates than DIASTAT(TM). The difference in the positive rates among four commercial ELISA kits may come from the different antigen sources. The panel test of 8 autoantibodies using Varelisa(TM) ELISA kit offers discriminative power and enhances the specificity of the assay in patients who lack clear evidence of clinically definite autoimmune disease.
Antibodies ; Autoantibodies* ; Autoimmune Diseases ; Diagnosis ; Diagnosis, Differential ; Enzyme-Linked Immunosorbent Assay* ; Histidine-tRNA Ligase ; Humans ; Rheumatic Diseases ; Sensitivity and Specificity ; Staphylococcal Protein A

The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.
Antibodies ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Histidine-tRNA Ligase ; biosynthesis ; genetics ; immunology ; Humans ; Open Reading Frames ; genetics ; Prokaryotic Cells ; metabolism

Although corticosteroids have been the initial agent for the treatment of inflammatory myopathies (IM), immunosuppressive agents such as azathioprine, methotrexate, cyclophosphamide, or cyclosporine are commonly required to control the disease except mild cases. On the other hand, the efficacy of combination therapy of cyclosporine and methotrexate in severe rheumatoid arthritis has been proven without serious side effects. However, in treatment-resistant myositis, the experience of such a therapy is very limited, and has not been described in refractory polymyositis with anti-Jo-1 antibody. Here, we report a young female patient with recalcitrant polymyositis and anti-Jo-1 antibody who was successfully treated with the combination therapy of cyclosporine and methotrexate. At first, the myositis did not respond to several agents, such as corticosteroid, monthly pulse cyclophosphamide, azathioprine, or cyclosporine. Methotrexate was initially avoided as treatment regimen because of its potential pulmonary toxicity in the case with preexisting lung disease.
Adult ; Antibodies, Antinuclear/blood* ; Autoantigens/immunology ; Cyclosporine/administration & dosage ; Cyclosporine/therapeutic use* ; Drug Resistance ; Drug Therapy, Combination ; Female ; Histidine-tRNA Ligase/immunology ; Human ; Immunosuppressive Agents/administration & dosage ; Immunosuppressive Agents/therapeutic use* ; Methotrexate/administration & dosage ; Methotrexate/therapeutic use* ; Polymyositis/drug therapy* ; Polymyositis/immunology