两种酶标抗体制备方法的比较及李斯特菌TASELISA试剂盒性能的优化  

Comparison of two methods in preparation of enzyme conjugates and optimization of Listeria monocytogenes TAS-ELISA KIT

作　　者：刘雅莉[1,2,3] 刘芳 刘箐[3] 韩舜愈[2] 孙志博[5] 夏俊芳[2] 朱小清 

机构地区：[1]兰州大学第二医院,甘肃兰州730030 [2]甘肃农业大学食品科学与工程学院,甘肃兰州730070 [3]上海理工大学医疗器械与食品学院,上海200093 [4]甘肃出入境检验检疫局国际旅行卫生保健中心,甘肃兰州730020 [5]兰州大学基础医学院,甘肃兰州730000 [6]甘肃出入境检验检疫局,甘肃兰州730020

出　　处：《食品工业科技》2012年第15期301-306,310,共7页Science and Technology of Food Industry

基　　金：质检总局科研项目(GSCIQ_2010IK220)

摘　　要：研制一种低成本、快速、准确的三抗体夹心酶联免疫(Three antibodies sandwich ELISA,TAS-ELISA)检测试剂盒。采用戊二醛法、改良过碘酸钠法分别制备碱性磷酸酶(Alkaline Phosphatase,AP)、辣根过氧化物酶(Horseradish Peroxidase,HRP)标记的山羊抗兔IgG,比较单核细胞增生性李斯特菌(Listeria monocytogenes,LM)TAS-ELISA试剂盒两种酶标抗体的检测效果,并考察了试剂盒的检测灵敏度、特异性、稳定性、保存周期等指标。结果表明,采用改良过碘酸钠法制备的HRP-IgG效果好,克分子比最高可达2.9,酶结合率达27%;试剂盒检测周期6h,检测成本为3.5元/孔,特异性实验、干扰实验、重复性实验、稳定性实验表明采用该方法制备的酶标抗体,特异性好、结果稳定可靠。自制TAS-ELISA试剂盒检测性能可达到商业化试剂盒水平,为该产品的产业化提供了一定的理论依据。To develop a low-cost,rapid and accurate TAS-ELISA KIT.This study preparated AP-IgG and HRP-IgG by glutaraldehyde method and improved periodate method respectively and compared two enzyme conjugates of Listeria monocytogenes TAS-ELISA KIT,further investigated some indexes including the sensitivity,specificity,stability,shelf life.The results showed that HRP-IgG preparated with improved periodate method was more effective,the molar ratios was 2.9,the rate of enzyme-labeled was 27%.The detection time of KIT was 6h,the detection cost was $3.5/well.Specific experiment,interference experiment,repetitive experiment and stable experiment indicated that the HRP-IgG preparated with improved periodate method was specific and reliable.The performance of self-made TAS-ELISA KIT could reach commercial KIT,it also could provide theoretical basis for industrialization of the product.

关 键 词：单核细胞增生性李斯特菌 改良过碘酸钠法 碱性磷酸酶 辣根过氧化物酶 三抗体夹心ELISA 

分 类 号：TS207.3[轻工技术与工程—食品科学]