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本研究旨在建立人IFN-γ体外释放检测法,用于人结核病的特异性诊断。克隆表达了人IFN-γ基因,利用纯化的重组IFN-γ免疫小鼠,获得两株高效价的单克隆抗体。用所获得的单克隆抗体及兔抗IFN-γ多克隆抗体建立了检测人IFN-γ的夹心ELISA,检测灵敏度达到31.25pg/mL。采集111位结核病阳性病人与292位临床健康对照者肝素抗凝全血,利用结核菌特异性抗原ESAT-6/CFP-10融合蛋白体外刺激外周血淋巴细胞释放IFN-γ,用所建立的夹心ELISA及商品化试剂盒平行检测所有样本,结果表明两种方法的检测结果相符。结核患者的检测灵敏度为95.5%,健康对照的阳性检出率为16.7%,患者与健康对照的阳性检出率差异极显著(P<0.01),证实所建立的方法灵敏度与特异性均很高,具有良好的应用前景。
This study aimed to establish a human IFN-γ in vitro release assay for the specific diagnosis of human tuberculosis. Human IFN-γ gene was cloned and expressed, and two high titer monoclonal antibodies were obtained by immunizing mice with purified recombinant IFN-γ. A sandwich ELISA for detecting human IFN-γ was established using the obtained monoclonal antibody and rabbit anti-IFN-γ polyclonal antibody with a detection sensitivity of 31.25 pg / mL. Collect 111 heparin anticoagulant whole blood from 111 tuberculosis positive patients and 292 healthy controls and stimulate the release of IFN-γ from peripheral blood lymphocytes by ESAT-6 / CFP-10 fusion protein of tuberculosis-specific antigen. ELISA and commercial kits parallel test of all samples, the results show that the two methods of testing results. The detection sensitivity was 95.5% for tuberculosis patients and 16.7% for healthy controls. The positive detection rate was significantly different between patients and healthy controls (P <0.01), confirming the high sensitivity and specificity of the established method , Has a good application prospects.