结核分枝杆菌融合蛋白ELISA方法检测
Evaluation of Mycobacterium tuberculosis fusion protein with ELISA in serological diagnosis
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摘要: 目的 探讨融合蛋白ESAT6-38kDa-16kDa对结核病的血清学诊断价值,并与脂阿拉伯甘露糖抗原(LAM)及结核蛋白芯片方法的检测结果进行比较,评价其应用价值。方法 表达、纯化重组目的蛋白,分别以此蛋白和LAM为抗原包板,以酶标抗体为二抗,采用ELISA间接法检测肺结核患者血清、肺外结核患者血清及正常人血清,比较敏感性、特异性等,评价其对结核病的诊断价值。结果 融合蛋白ESAT6-38kDa-16kDa、LAM、血清及酶标抗体的最佳工作稀释度分别为1、0.5μg/mL,1:100、1:4 000;分别用ESAT6-38kDa-16kDa和LAM检测761份结核病人血清,敏感性分别为76.9%、77.4%,特异性为89.2%、89.5%,差异均无统计学意义;ELISA法检测68份肺结核病血清标本融合蛋白,阳性检出率和阴性符合率与芯片检测结果有极高一致性。结论 融合蛋白ESAT6-38kDa-16kDa作为抗原对结核病血清学诊断具有一定应用价值。Abstract: Objective To study on applied value of recombinant ESAT6-38kDa-16kDa fusion protein in serological diagnosis of tuberculosis(TB) and to compare the results to imported protein lipoarabinomannan(LAM) and tuberculosis protein chip system.Methods Using ESAT6-38kDa-16kDa fusion protein and imported LAM protein as antigen and staphylococcal protein A linked to horseradish peroxidase (SPA-HRP) as the antibody,the serum of pulmonary tuberculosis patients,extrapulmonary tuberculosis patients and normal human were detected with indirect enzyme-linked immunosorbent assay(ELISA) then the value in serodiagnosis of TB were assessed.Results The indirect ELISA method for the detection of TB specific antibody in serum was established,with the optional antigen concentrations of 1 μg/mL and 0.5 μg/mL for ESAT6-38kDa-16kDa and ManLAM,and optimal serum and SPA-HRP dilution of 1:100 and 1:4000.The sensitivity and specificity of the assay were 76.9% and 89.2%,respectively.There was no significant difference in comparison with the results of LAM (P > 0.05).There was no significant difference in the positive and negative coincidence rate of the method in comparison with tuberculosis protein chip system based on the detection of 68 serum samples of pulmonary tubercuosis patients(χ2=0.018,P > 0.05).Conclusion This recombinant fusion protein may be used as an effective antigen in serodiagnosis of TB and helpful in developing new specific diagnostic test.
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