摘要： 目的·研制适用于检测钩端螺旋体（钩体）特异性抗体的间接酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）检测试剂盒，并对其性能进行评估。方法·选择我国主要致病性钩体流行株中高度保守的外膜蛋白进行原核表达，将获得的重组蛋白纯化后作为包被抗原行质谱鉴定，并对其抗原性进行检测。选用40例经确诊的钩体病患者血清、50例其他感染类疾病患者血清和391例健康对照者的血清样本作为检测样本，用钩体病确诊患者血清制备试剂盒内控阳性对照品和参考品、高效价混合阳性血清，用健康对照者血清制备试剂盒内控阴性对照品和参考品，用高效价混合阳性血清制备试剂盒内控弱阳性对照品（临界参考品）及灵敏度参考品，从钩体病确诊患者血清中选择强阳性、弱阳性血清（各1份）分别作为试剂盒内控强阳性、弱阳性精密度参考品。建立间接ELISA试剂盒检测体系，优化其制备工艺及反应条件，并对该试剂盒的精密度、敏感性、特异性和稳定性等性能进行评估。结果·钩体重组外膜蛋白纯化后经质谱鉴定为目标蛋白，其作为包被抗原具有较强的抗原性。经性能评估显示，间接ELISA试剂盒的批内精密度<15%、批间精密度<20%，其敏感性为95.0%、特异性为95.0%，且在2~8 ℃下储存12个月后性能仍然稳定。结论·以钩体重组蛋白为包被抗原的间接ELISA钩体抗体检测试剂盒的特异性、敏感性及其他性能指标均符合体外检测试剂盒性能要求，适用于临床辅助诊断、钩体病哨点监测及流行病学调查。
关键词: 钩端螺旋体, 重组蛋白, 间接酶联免疫吸附测定, IgG抗体, 敏感度/特异度 Abstract: Objective·To develop an indirect enzyme-linked immunosorbent assay (ELISA) kit for the detection of leptospira-specific antibodies and evaluate its performance.
Methods·The highly conserved outer membrane protein in the epidemic strains of the main pathogenic leptospira in China was selected and expressed by prokaryotic expression system. The recombinant protein was purified and used as coating antigen for mass spectrometry identification, and its antigenicity was detected. Serum samples from 40 patients with leptospirosis, 50 patients with other infectious diseases and 391 healthy controls were selected as test samples. The positive control, positive reference and high-titer mixed positive serum for internal control of the kit were prepared from the serum of patients with leptospirosis, the negative control and reference for internal control of the kit were prepared from the serum of healthy controls, and the weak positive control (critical reference) and sensitivity reference for internal control of the kit were prepared from high-titer mixed positive serum. One strong positive serum and one weak positive serum selected from the serum of patients with leptospirosis were used as strong positive and weak positive precision reference for internal control of the kit. The detection system of the indirect ELISA kit was established, its preparation process and reaction conditions were optimized, and its precision, sensitivity, specificity and stability were evaluated.
Results·The purified recombinant outer membrane protein of leptospira was identified as the target protein by mass spectrometry. The results of performance evaluation showed that the intra assay precision and inter assay precision of the indirect ELISA kit were less than 15% and 20%, respectively, and the sensitivity and specificity were 95.0% and 95.0%, respectively. The performance of indirect ELISA kit was still stable after 12 months' storage at 2?8 ℃.
Conclusion·The specificity, sensitivity and other performance indicators of the leptospira antibody detection kit meet the requirements of the in vitro test kit, which may be suitable for clinical auxiliary diagnosis, sentinel monitoring of leptospirosis and epidemiological investigation.
Key words: leptospira, recombinant protein, indirect enzyme-linked immunosorbent assay, IgG antibody, sensitivity/specificity PDF全文下载地址:
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