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1、HYBRIDOMAANDHYBRIDOMICSVolume23,Number2,2004©MaryAnnLiebert,Inc.MonoclonalAntibodiesMurineMonoclonalAntibodytotheSARSCoronavirus(SARSCoV)SpikeProteinANTIGENUSEDFORIMMUNIZATIONCloneClassWeusedinactivatedwholeSARSCoV(coronavirus)asanti-F26G1IgG2a/kgen(courtesyofDr.AntonAndonov,NationalMicrobiologyF26
2、G6IgG2b/kLaboratory,Winnipeg,Canada).F26G8IgG2a/kF26G18aIgG2b/kF26G19aIgG2a/kMETHODOFIMMUNIZATIONSPECIFICITYForimmunizations,5–6-week-oldfemaleBALB/Cmicewereinjectedsubcutaneouslywith50mgofinactivatedSARS-CoVSARS-CoVspikeproteinwithanequalpartofCompleteFreund’sAdjuvant(CFA,H37Ra;Difco),onday1.Onday
3、30themicereceived50mgofpurifiedSARS-CoVinIncompleteFreund’sAdjuvant(IFA).SOURCEOndays48and63,micereceived5mginIFA.Themicere-ceivedafinalboosterinjectionwith5mgofpurifiedSARSMurinevirus3dayspriortofusion.Micewereeuthanisedbyanaes-thesiaoverdoseandexsanguinatedbycardiacpuncture.Thespleenswereexcisedu
4、nderasepticconditions.FORMAT/PURITYSupernatantsPARENTALCELLLINEUSEDIMMUNOGENFORFUSIONWholeinactivatedTOR-3SARSCoVP3X63Ag8.653ANTIGENREACTIVITYSELECTIONANDCLONINGEIA,Western,IFA,IHCOFHYBRIDOMASImmunizationofmice,removalofspleens,preparationofAPPLICATIONSspleenandmyelomacells,fusionandscreeningformon
5、oclonalantibodytoSARSwereperformedaccordingtoNCFADstan-EIA,IHC,Western,IFA,cELISA,VNadardoperatingproceduresunderISO17025.Hybridomaswereclonedoutinsemisolidmedium.SupernatantswerescreenedviaELISAusingpurifiedvirusasantigen.Isotypingwasper-METHODOFIMMUNIZATIONformedusingacommercialdipsticktest(Roche
6、)accordingtothemanufacturer’sinstructions.s.q.137138MONOCLONALANTIBODIESSTORAGEADDRESSCORRESPONDENCETO:Frozen,220Dr.JodyD.Berry,XinYuan,BrigitteNicolas,FranRanada,andFrankPlummerTheMonoclonalAntibodySectionMEDIANationalCentreforForeignAnimalDisease-CFIANationalMicrobiologyLaboratory–HealthCanadaBDC
7、ellQuantumYieldReagentDevelopmentUnit1015ArlingtonSt.AVAILABILITYWinnipeg,MB,CanadaTelephone:204-789-6063SupernatantYes3NoFax:204-789-2038E-mail:berryjd@inspection.gc.ca
