摘要
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 human IFN-γ （hlFN-γ） in vitro release assay and to apply it in diagnosis of human tuberculosis. Human IFN-γ gene was cloned and expressed in Escerichia coli. The recombinant hlFN-γ was purified and used as immunogen to immunize mice and rabbits respectively. Monoclonal and polyclonal antibodies were respectively developed ,and a sandwich ELISA was established. The heparized whole blood from 111 active tuberculosis patients and 292 clinical healthy controls were collected. The blood was stimulated with tuberculosis specific fused antigen ESAT-6/CFP-10 and the plasma was collected for IFN-γ detection. The sensitivity for tuberculosis diagnosis was 95.5%, whereas the positive detection rate for the healthy controls was 16.7%. There was a significant difference between the patients and healthy controls （P〈0.01） indicating that this assay had a high sensitivity and specificity, and thus could be promising in tuberculosis diagnosis.
出处
《生物工程学报》
CAS
CSCD
北大核心
2008年第9期1653-1657,共5页
Chinese Journal of Biotechnology
基金
国家973项目(No.2006CB504401)
武汉市科技攻关课题(No.20066002056)
国家“十一五”科技攻关奶业重大专项(Nos.2006BAD04A05,2006BAD04A12)资助~~
