摘要
目的:利用ELISA技术研发一种灵敏、快速检测人血清中脑红蛋白(Ngb)含量的检测试剂盒,并探讨其在正常人血清样本检测中的应用。方法:采用热诱导的原核表达体系获得重组人源脑红蛋白(rhNgb),通过凝胶过滤和阴离子交换层析等制备rhNgb纯品;将适量rhNgb纯品免疫动物获得抗rhNgb单克隆抗体和多克隆抗体;用双抗体夹心ELISA技术制备rhNgb-ELISA检测试剂盒;用该试剂盒对410例正常人血清中Ngb的含量进行检测,其中包括1.5岁以下婴儿血清55例、19～70岁成人血清355例。结果:用制备的rhNgb-ELISA试剂盒检测发现Ngb在正常人血清中的分布与年龄存在明显的相关性,表现为"两头高、中间低"的趋势,婴儿和老年人血清中Ngb的含量分别为1.6708±0.4945和2.1962±0.6703μmol/L,而中年人血清中Ngb的含量为0.3622±0.0716μmol/L。结论:采用双抗夹心ELISA技术并结合严格的质量控制,研发了高效灵敏的rhNgb-ELISA检测试剂盒,并初步获得了正常人血清中Ngb的含量分布情况,为临床缺血及缺氧性脑损伤等相关疾病的辅助诊断治疗提供了参考信息。
Objective：To develop an ELISA kit of human neuroglobin（Ngb） using sandwich technique and to determine the distribution of Ngb normal human serum.Methods：The recombinant human Ngb（rhNgb）（also used as antigen） was expressed by thermal-induced and purified by gel filtration and ion exchange chromatography.The animals were immunized by purified rhNgb to obtain the anti-Ngb monoclonal antibody and polyclonal antibody.Finally,the ELSIA kit was prepared by sandwich technique,and this sandwich ELISA kit was used to detect the distribution of Ngb in 410 cases of normal human serum,including 55 cases of infants（age〈1.5） and 355 cases of adults（age as 19～70）.Results：Our data showed that the Ngb protein in normal human serum was associated with the age and showing a pattern of ″higher in young and elder persons and lower in middle age ones″.The content of Ngb in infants and olders were 1.6708±0.4945 and 2.1962±0.6703 μmol/L,while in middle-aged was 0.3622±0.0716 μmol/L.Conclusion：The double-antibody sandwich technique was used to prepare a high-efficient and sensitive Ngb-ELISA kit and further the kit was used to detect the distribution of Ngb in normal human serum.It will provide a reference for auxiliary diagnosis and treatment of clinical ischemia-hypoxic related diseases
出处
《生物技术通讯》
CAS
2011年第1期28-31,44,共5页
Letters in Biotechnology
基金
国家自然科学基金(30973107
30772293)
国家重点基础研究发展计划(2006CB504100)
国家科技重大专项(2009ZX09503-002
2009ZX09301-002
2009ZX09103-616)
北京市科委科技攻关计划(Z07000200540702)
