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鸭坦布苏病毒囊膜E蛋白单克隆抗体的制备及ELISA检测方法的建立.docx

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鸭坦布苏病毒囊膜E蛋白单克隆抗体的制备及ELISA检测方法的建立 Title:PreparationofMonoclonalAntibodiesagainstDuckTembusuVirusEnvelope(E)proteinandEstablishmentofELISADetectionMethods Abstract: DuckTembusuvirus(DTMUV)isanimportantpathogenthatcausessignificanteconomiclossesintheduckindustry.Theenvelope(E)proteinofDTMUVplaysacriticalroleinviralreplicationandisapotentialdiagnostictarget.Inthisstudy,weaimedtodevelopmonoclonalantibodies(mAbs)againstDTMUVEproteinandestablishanenzyme-linkedimmunosorbentassay(ELISA)forthedetectionofDTMUVinfection. Introduction: DTMUVisaflavivirusthatprimarilyaffectsducksandcausessymptomssuchasreducedeggproduction,neurologicaldisorder,andmortalityininfectedbirds.ThetimelyandaccuratediagnosisofDTMUVinfectioniscrucialfordiseasecontrolandprevention.TheenvelopeproteinofDTMUVishighlyimmunogenicandisknowntoinduceprotectiveimmuneresponses.Therefore,developingspecificantibodiesagainstDTMUVEproteincanaidintherapidandsensitivediagnosisofDTMUVinfection.TheELISAdetectionmethodisawidelyusedtechniqueforthediagnosisofviralinfectionsduetoitshighsensitivityandspecificity. Methods: 1.ProductionofDTMUVEprotein: -DTMUVEgenewasclonedandexpressedinasuitableexpressionsystem. -RecombinantEproteinwaspurifiedusingaffinitychromatographyandconfirmedbySDS-PAGEandWesternblotanalysis. 2.Immunizationofmiceandproductionofhybridomas: -BALB/cmicewereimmunizedwithpurifiedDTMUVEproteininthepresenceofanadjuvant. -Spleencellsfromimmunizedmicewerefusedwithmyelomacellstogeneratehybridomas. -HybridomacellswerescreenedfortheproductionofantibodiesagainstDTMUVEproteinusingindirectELISA. 3.Selectionandexpansionofspecifichybridomaclones: -Positivehybridomasweresubclonedbylimitingdilutiontoensuremonoclonality. -Theselectedspecifichybridomacloneswereexpandedinculture. 4.Characterizationofmonoclonalantibodies: -Culturesupernatantsofspecifichybridomacloneswerecollected,andmAbswerepurified. -ThespecificityofmAbswasdeterminedbyWesternblot,immunofluorescenceassay,andneutralizationassay. 5.EstablishmentofELISAforDTMUVdetectio