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当前位置: 首页 > 文章 > 间接竞争性ELISA法快速诊断TGEV的研究 中国兽医学报 2012,32 (11) 1615-1623
Position: Home > Articles > Detection of TGEV with indirect competitive ELISA Chinese Journal of Veterinary Science 2012,32 (11) 1615-1623

间接竞争性ELISA法快速诊断TGEV的研究

作  者：
李丹丹;朱振营;徐义刚;刘志强;高慎阳;李一经
单  位：
中国检验检疫科学研究院动植物检疫研究所;海南出入境检验检疫局检验检疫技术中心;黑龙江出入境检验检疫局检验检疫技术中心;辽宁医学院畜牧兽医学院;东北农业大学动医学院
关键词：
TGEV;单克隆抗体;间接竞争性ELISA
摘  要：
利用基因工程技术制备的猪传染性胃肠炎病毒(TGEV)N基因原核表达产物重组N蛋白作为抗原诊断试剂,抗重组N蛋白的单克隆抗体作为抗体诊断试剂建立了间接竞争性ELISA检测细胞培养物中的TGEV病原的方法,并确定了间接竞争性ELISA操作流程中的最佳反应条件。在间接竞争性ELISA中,最佳抗原包被量为0.25μg/孔;竞争抗原与单克隆抗体作用的最适时间为1h,反应温度为37℃;选择1h作为酶标抗体作用的最佳时间;底物液最佳作用时间为10min;选择样品的抑制率50%为其临界值;所用封闭液0.5%的聚乙烯醇PBS溶液在4℃冰箱中可密封保存6个月,封闭时间为120min;特异性试验表明与猪轮状病毒、猪流行性腹泻病毒等肠道腹泻性病毒均无交叉反应。本试验建立的间接竞争性ELISA诊断方法具有良好的敏感性和特异性,为TGEV的疫情监测、及时而准确的诊断奠定了基础。
译  名：
Detection of TGEV with indirect competitive ELISA
作  者：
LI Dan-dan1,2,ZHU Zhen-ying3,XU Yi-gang4,LIU Zhi-qiang1,GAO Shen-yang5,LI Yi-jing2(1.Animal Quarantine Lab,Inspection&Quarantine Technology Center of Hainan Entry-Exit Inspection & Quarantine Bureau,Haikou 570311,China;2.College of Veterinary Medicine Northeast Agriculture University,Harbin 150030,China;3.Institute of Animal and Plant Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100029,China;4.Animal Quarantine Lab,Inspection & Quarantine Technology Center of Heilongjiang Entry-Exit Inspection & Quarantine Bureau,Harbin 150001,China;5.Department of Animal Husbandry & Veterinary Medicine,Liaoning Medical University,Jinzhou,Liaoning 121001,China)
关键词：
TGEV;monoclonal antibody;indirect competitive ELISA
摘  要：
Three methods were established for rapid detection to TGEV using TGEV recombinant nucleoprotein(rNP)and McAb against rNP.In indirect competitive ELISA the optimal amount of coated antigen was 0.25 μg per well;the optimal time in which McAb reacted with competitive antigen was 1 h and the reaction temperature was 37℃;the reaction time to HRP was 1 h;the reaction time to substrate was 10 min;the inhibited rate of 50% was the critical value.The optimal blocking solution was 0.5% polyvinyl alcohol(PVA) of PBS,the blocking time was 120 min;blocking solution can be kept for 6 monthes at 4℃.The special test indicates TGEV had no crossing reaction with PRV and PEDV.The method for detection was rapid,sensitive,accurate and simple.The result would be gained in 2-3 hours.The method for rapid detection to TGEV was established firstly by using rNp and McAb against rNP in domestic,and founded a material base for TGE detection accurately and rapidly.

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