西尼罗河病毒抗体竞争elisa检测方法的建立与初步应用

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1、扬州大学硕士学位论文n一选出特异性强的2株单抗(8F4A4，6H381)，为建立能够鉴别诊断JEV抗体和WNV抗体的竞争ELISA方法提供了竞争检测抗体。以纯化的WNV囊膜蛋白E结构域III作为抗原包被ELISA反应板，单抗8F4A4作为竞争检测抗体，通过方阵试验确定了重组EIII蛋白抗原的最佳包被浓度和单抗最佳稀释度，同时改变血清稀释倍数、包被条件及竞争反应时间、封闭液、酶标二抗稀释度及作用时间、底物最佳作用条件等因素，确定了最佳反应条件。结果表明：重组蛋白最佳包被浓度为530ng／mL，单抗的最佳稀释倍数为1：8000，待检血清的最佳稀释倍

2、数为l：10。本研究在国内首次建立了检测WNV抗体的竞争ELISA方法，特异性试验证实该方法能够与我国流行的JEV进行鉴别诊断。对临床56份阴性样品进行检测计算出该方法的临界值为30％，以此为判定标准对送检的711份血清进行检测，结果均为阴性。此方法的建立弥补了商品化试剂盒不能区分JEV和WNV感染的缺陷，为我国西尼罗河病毒病的流行病学调查提供了一种有效的抗体检测方法。关键词：西尼罗河病毒；囊膜蛋白E；蛋白表达：竞争ELISA常娓娓：西尼罗河病毒抗体竞争ELISA检测方法的建立及初步应用DevelopmentandApplicationofaC

3、ompetitiveEnzyme--LinkedImmunosorbentAssayDetectingAntibodyagainstWestNileVirusMasterCandidate：WeiweiChangAdvisor：Prof．ZhiliangWangProf．XiufanLiuAbstractWNVandJEVarethemembersoftheJapaneseencephalitisvirusgroupofthegenusFlavivirusfamilyFlaviviridae．Thesevirusesaremosquito—bo

4、rneflavivirus，andmaintainedinbird—mosquito-birdcycleinnature，withhumansandothervertebrateanimalslikehorseasaccidentalhosts．WNVwasfirstisolatedin1937fromthebloodofafebrilefemalepatientintheWestNiledistrictofUganda，thenitoutbrokeinNewY．orkin1999whichcausedpeoplepanicandgreatec

5、onomiclosses．OfficeInternationalDesEpizooties(OIE)classifiedthisdiseaseasnotifibledisease．ChinawasJEVepidemicdistrictandJEVhasRologycrossreactionwithWNVinordertOexcludesuspectedCaSeSofⅥ仓『、(SOwearenecessarytoestablishadifferentialdiagnosismethodsastechnicalreserves．Envelopepr

6、oteinisthemostimportantstructuralproteinofWNV，itsdomainIIIcontainsantigenepitopeswhichspecifictOWNVanditsmainneutralizingepitopesarelocatedintheE307、E330、E332residues．thisareahasalwaysbeenconsideredasreceptorarea．Throughtherecognitionofneutralizationantibodyofthisarea，wecand

7、istinguishbetweenJapaneseencephalitisvirus(厄v)，yellowfevervirus，denguevirusandMurrayvalleyencephalitisvirus(MVEV)．AccordingtothepublishedsequenceoftheWNVNY99strainandJEV一一一一．堑型奎兰堕主堂垡鲨茎竖SA14．14．2strain，onepairofspecificprimersforWNVEproteindomainIIIandJEVEproteindomain111were

8、designedrespectively，andthedomainIIIgeneofWNVandJEVwereamplifiedbypolymeras