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1、HYBRIDOMAANDHYBRIDOMICSVolume23,Number2,2004©MaryAnnLiebert,Inc.MonoclonalAntibodiesF26G15:MurineMonoclonalAntibodytotheSARSCoronavirusNucleoprotein(SARSCoV)ANTIGENUSEDFORIMMUNIZATIONISOTYPEWeusedinactivatedwholeSARSCoV(coronavirus)asanti-IgG2b/kgen(courtesyofDr.
2、AntonAndonov,NationalMicrobiologyLaboratory,Winnipeg,Canada).SOURCEMETHODOFIMMUNIZATIONMurineForimmunizations,5–6-week-oldfemaleBALB/Cmicewereinjectedsubcutaneouslywith50mgofinactivatedSARS-CoVwithanequalpartofCompleteFreund’sAdjuvant(CFA,H37FORMAT/PURITYRa;Difco
3、),onday1.Onday30themicereceived50mgofpurifiedSARS-CoVinIncompleteFreund’sAdjuvant(IFA).SupernatantsOndays48and63,micereceived5mginIFA.Themicere-ceivedafinalboosterinjectionwith5mgofpurifiedSARSvirus3dayspriortofusion.Micewereeuthanisedbyanaes-thesiaoverdoseandexs
4、anguinatedbycardiacpuncture.TheIMMUNOGENspleenswereexcisedunderasepticconditions.WholeinactivatedTOR-3SARSCoVPARENTALCELLLINEUSEDFORFUSIONANTIGENREACTIVITYP3X63Ag8.653EIA,WesternSELECTIONANDCLONINGOFHYBRIDOMASAPPLICATIONSImmunizationofmice,removalofspleens,prepar
5、ationofEIA,Western,cELISAspleenandmyelomacells,fusionandscreeningformonoclonalantibodytoSARSwereperformedaccordingtoNCFADstan-dardoperatingproceduresunderISO17025.Hybridomaswereclonedoutinsemisolidmedium.SupernatantswerescreenedMETHODOFIMMUNIZATIONviaELISAusingpu
6、rifiedvirusasantigen.Isotypingwasper-formedusingacommercialdipsticktest(Roche)accordingtos.q.themanufacturer’sinstructions.SPECIFICITYSTORAGENucleoproteinFrozen,220139140MONOCLONALANTIBODIESMEDIAADDRESSCORRESPONDENCETO:Dr.JodyD.Berry,XinYuan,BrigitteNicolas,FranR
7、anada,BDCellQuantumYieldandFrankPlummerTheMonoclonalAntibodySectionNationalCentreforForeignAnimalDisease-CFIANationalMicrobiologyLaboratory–HealthCanadaAVAILABILITYReagentDevelopmentUnit1015ArlingtonSt.SupernatantYes3NoWinnipeg,MB,CanadaTelephone:204-789-6063Fax:
8、204-789-2038E-mail:berryjd@inspection.gc.ca
