An ELISA for detection of anti-dsDNA by using plasmid DNA as antigen / 中华检验医学杂志

ABSTRACT

Objective To develop an ELISA for detection of anti-dsDNA antibody by using plasmid DNA as antigen.Methods DNA in plasmid pBV220 for prokaryotic expression vector was purified by base-cleavage. The microtiterplates were pretreated by poly-L-lysine and coated by the plasmid DNA in a dilution of 1∶50 as antigen. An ELISA method for detection of serum anti-dsDNA antibodies was developed with HRP-SPA as enzyme-labeled marker.The IIF using crithidia lucilia as substrate was performed simultaneously for comparison. The serum samples from 64 patients with SLE, 8 with MCTD and 17 with RA were detected. Results The concentration of DNA was 1 54 g/L by UV spectrophotometer at wavelength of 260 nm. The positive percentage of ELISA for anti-dsDNA was higher than that of IIF. By comparison with IIF the positive percentages in SLE, MCTD and RA groups were 23 4% vs 17 2%, 12 5% vs 12 5% and 11 8% vs 5 9%, respectively, and the coincident rates between the 2 methods were 93 8%, 100% and 94 1% respectively. The sensitivity and specificity of the developed ELISA for detection of anti-dsDNA were 100% and 93 4% when IIF was as gold standard.Conclusion The ELISA by using plasmid DNA as antigen to detect anti-dsDNA has fine precision, sensitivity and specificity. Its positive rate is higher than that of IIF thus it will contribute to monitor the activities for SLE patients′ condition.