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	Theproductionofmonoclonalantibodybyhybridomafusion:ImmortalizationofsensitizedBlymphocytesfromimmunemice.

	Overview
	Weoutlineasimpleandcontemporaryprotocolforthedevelopmentofmonoclonalantibodiesusinghybridomafusioninimmunemice(1).Whilethebasicstyleofthisfusionissimilartoothers(2,3),thisprotocolhasseveralsubtlebutsignificantmodifications.Theseincludetheuseof:spleenperfusionratherthancrushingtoseparatethespleencells(whichreducestheamountofcontaminatingfibroblastandlipoidalmaterial);commercialHybridomaCLoningFactors(ratherthanfeederlayers);commerciallypreparedsemi-solidHATcontainingagarose,ratherthanlimitingdilution.Elementsofthisprotocolspanseveralresearchinstitutionsandmanyyearsofexperience(4,5,6).

	Material
	Sterile10,25,and50mlSEROlogicalPipettes,Pipet-Aid,15and50mlcentrifugetubes(Falconsterile),Tissuecultureflasks(25cm2,75cm2and125cm2),indelIBLewaterproofMarker.Sterile1mlpipettetipsforGilsonp1000pipetteman.370CwaterbathandThermometer.Humidified370C,5%CO2tissueculturecABInet.ClassIIBIOLOGicalSafetyCabinet.InvertedMicroscope.Benchtopcentrifugecontaininga4swingingbucketrotor,atroomtemperature.Stopwatchortimer.Multichannelpipettorandappropriatesteriletips,steriledisposablepetridishes.Sterile96-wellflat-bottomedcellcultureplates.Reagents:1-3x108-9immunespleencells1-6x107-8myelomacellsinlogphaseofgrowthCompleteMediaNoSera(CMNS)forwashingofthemyelomaandspleencells.HybridomamediumCM-HAT{CellMab(BD),10%FBS(orHS);5%OrigenHCF(hybridomacloningfactor)containing4mML-glutamineandantibiotics}tobeusedforplatinghybridomasafterthefusion.HybridomamediumCM-HT(NOAMINOPTERIN){CellMab(BD),10%FBS5%OrigenHCFcontaining4mML-glutamineandantibiotics}tobeusedforfusionmaintenancestoredintherefrigeratorat4-60C.feedingfusionsondays4,8,and12,andsubsequentpassages.Thawedinactivatedandpre-filteredcommercialFetalBovineserum(FBS)orHorseSerum(HS)storedintherefrigeratorat40C.Mustbepretestedformyelomagrowthfromsinglecells.L-glutamine,200mM,100Xsolutionstoredat-200Cfreezer.TheL-glnisthawedandwarmeduntilcompletelyinsolution.TheL-glnisdispensedintomediatosupplementgrowth.L-glnisaddedto2mMformyelomas,and4mMforhybridomamedia.Penicillin,Streptomycin,Amphotericin(antibacterial-antifungal)isstoredat-200CuntilneededanditisthawedandaddedtoCellMabMediato1%.CellMabMedia,QuantumYieldfromBDisstoredintherefrigeratorat40Cinthedark.MyelomagrowthmediaisCellMabMediawithaddedL-glnto2mMandantibiotic/antimycoticsolutionto1%andiscalledCMNS.Donotaddantibioticstomediawhengrowingmyelomasforstock,onlyforpre-fusiongrowth.Indicatepresenceorabsenceofeachreagentonthebottlelabel.DonotadjustthepHasitcontainsHEPESbiologicalbufferalready.1bottleofPEG1500inHepes(Roche)(usefreshbottlethathasneverbeenopened)8-AzaguanineisstoredasthedriedpowdersuppliedbySIGMAat-700Cuntilneeded.Reconstitute1vial/500mlofmediaandaddentirecontentsto500mlmedia(eg.2vials/litre).MyelomaMediaisCMwhichhas10%FBS(orHS)and8-Aza(1X)storedintherefrigeratorat40c.ClonalcellmediumD(Stemcell,Vancouver)containsHATandmethylcelluloseforsemi-soliddirectcloningfromthefusion.Thiscomesin90mlbottleswithaCoAandmustbe"meltedat37Ocinawaterbathinthemorningofthedayofthefusion.LoosenthecapandleaveinCO2incubatortosufficientlygasthemediumDandbringthepHdown.HybridomasupplementsHT[hypoxanthine,thymidine]aretobeusedinmediumforthesectionofhybridomasandmaintenaceofhybridomasthroughthecloningstagesrespectively.OrigenHCFcanbeobtaineddirectlyfromIgenandisacellsupernatantproducedfromamacrophage-likecell-line.Itcanbethawedandaliqoutedto15mltubesat5mlpertubeandstoredfrozenat-200C.PositiveHybridomasarefedHCFthroughthefirstsubcloningandaregraduallyweaned.Itisnotnecessarytocontinuetosupplementunlessyouhaveaparticularlydifficulthybridomaclone.Thisandotheradditiveshavebeenshowntobemoreeffectiveinpromotingnewhybridomagrowththanconventionalfeederlayers.

	Procedure
	Atleastoneweekpriortoexpectedfusiondatethawafreshvialofmyelomacells.Itisadvisabletokeepseveralflasksatdifferentdensitiessothatyoucanchoosethebestoneonthedayofthefusion.Wegenerallytrytouseaflaskthatisactivelydividingandatacelldensityof3-6x105cells/ml.Donotletthemovergrowortheywillenteradeclinephase.Twotofivedaysbeforethescheduledfusiongiveafinalinjectionof~5ugofantigeninPBSi.p.orintravenouslyintailveinofthemouse(withhightiteralreadydetermined).1.Spindownmyelomasandwashwith30mlserumfreemedia(CMNShasglutamine).Usetabletopcentrifugeat850rpmfor12minutes.Performviablecellcountwithtrypanblueexclusionprinciple,andwashcellswith30mlofRPMI-CMNS.Spindownasabove,resUSPendinCMNSanddisperse.Leaveat37°Cuntilspleensareretrieved.Testaminopterinsensitivity.Keep1millionmyelomacellsforcontrolplateandtransferintoa15mlconical.Todoso,add15mlofHATmediatothemillionmyelomacellsandplateout2drops/wellona96wellplate.2.Removespleenfrommouseinthebiohazardfacility.Euthanisethemiceandsubmergeitin70%ETOH.Letthemouseairdryonitsrightsideonapapertowel.Removespleenusingsterileinstrumentsandcarefullyputintolabeled10mlofRPMI-CMwithantibioticsand20%FCSfortransportbacktothelab.Disposeofmouseandleavefacility.3.Placespleenintosterilepetridishes.Add10mlofRP-I-CMNSandperfusethecellsoutofthespleen.Pokethespleen8-10timeswithan18ganeedle(holdwithsterileforceps).Usea21gaona3mlsyringetodrawupsomeRPMI.InjecttheRPMIslowlyintothespleenabout50-100timesuntilnearlyallthecellsarewashedout.Discardthespleensintothebiohazardsbag.4.Collectandtransferthespleencellstoanew50mlconicaltube.Rinseoutthedish2Xwith10mlofRPMI-CMNSandpoolwiththefirst10ml(theuseofperfusionremovestheproductionoflargedebrisseenwithgrinding,andobviatestheneedtoletthedebrissettle).Spindownat900rpmfor12minutes.Discardthesupernatanttobleachcontainer.Washthecellswithanother30mlRPMI-CMNS.Removeasmallsampleandcounttheviablecell/mlandspinagainasabove.Combinethecellsataratioof5:1(spleencells:myelomacells)andnever1X10myelomacells.5.Washboththemyelomaandspleencells2moretimeswith30mlofRPMI-CMNS.Spinat800rpmfor12minutes.6.Removesupernatantandresuspendcellsin5mlofRPMI-CMNSandpooltogether.Fillvolumeto30mlandspindownasbefore.7.Aspirateallfluidintobleachvessel.Breakuppelletbygentlytappingontheflowhoodsurface.Add1mlofBMBREG1500(prewarmedto37°C)dropwisewith1ccneedleover1minute.SwirlandtaptheconicalgentlywhileaddingthePEGtoresuspendthecells.8.Add1mlofRPMI-CMNStothePEGcellsgentlyover1minutewhileswirling(todilutethePEG).9.Add8mlRPMI-CMNSover2minutestoslowlydiluteoutthePEG.10.Incubatethecellsinthe37°Cwaterbathfor10minutes.Centrifugethecellsat700rpmfor10minutes(themembranesarestillveryweak).11.Aspirateallfluid,andadd5mlofRPMI-CM-10%FCSWITHOUTRESUSPENDINGTHECELLS!Thecellswilldisperseadequatelybysimplyaddingthemediaatthispoint.12.Incubate@37°Canother10minutes.(12.5Meanwhileputaside1mlofClonacellmediumDformyelomatesting.)13.Gentlydilutecellsin5mlofCompletemediaandtransferinto95mlofClonacellMediumD(HAT)media(with5mlofHCF)andplateout10mlpersmallpetriplate.14.Diluteabout1000P3X63Ag8.653myelomacellsinto1mlofmediuDandtransferintoasinglewellofa24wellplate.Thisisthemyeloma/HATcontrol.P15.Placeplatesinincubatortwoplatesinsideofalargepetriplate,withanadditionalpetriplatefullofdH20withoutalidforhumidity.Leavefor10-18daysunder5%CO2overlayat37degrees.15.Pickclonesfromsemisolidagaroseinto96wellplatescontaining150-200ulofCM-HT.Screensups4dayslaterinELISA.Movepositiveclonesupto24wellplates.16.Heavygrowthwillrequirechangingofthemediaatday8(+/-150ml).Shouldseemacroscopiccoloniesatthistime.AtthistimecandecreasetheHCFto0.5%(gradually-2%,then1%,then0.5%)inthecloningplates.17.Isotypeviasupernatantsandgrowupforascites/largeflaskproductionandfurtherfreezedown.

	Troubleshooting
	IstronglyreccommendtouseSouthernBiotechGoatanti-MouseIg(H+L)HRPChainsforscreeningsupernatants.WehavescreenedanddevelopedMabstovariousantigensandhavesurprisinglyfoundoutthatusingsomesecondaryreagentsfromseveralothermajorcompaniescanresultinweakornoreactivity.Keywords:monoclonalantibody;hybridoma;fusion

	Reference
	1)KohlerG,andC.MilsteinContinuousculturesoffusedcellssecretingantibodyofpredefinedspecificity.1975.Nature256:495-497.2)Lane,R.D.Ashortdurationpolyethyleneglycolfusiontechniqueforincreasingproductionofmonoclonalantibody-secretinghybridomas.1985.J.Immunol.Meth.81:223-228.3)Harlow,E.andD.Lane.Antibodies:Alaboratorymanual.ColdSpringHarbourLaboratoryPress.1988.4)Kubitz,D.TheScrippsResearchInstitute.LaJolla.PersonalCommunication.5)Zhong,G.,Berry,J.D.,andChoukri,S.(1996)MappingepitopesofChlamydiatrachomatisneutralizingmonoclonalantibodiesusingphagerandompeptidelibraries.J.Indust.Microbiol.Biotech.19,71-76.6)Berry,J.D.,Licea,A.,Popkov,M.,Cortez,X.,Fuller,R.,Elia,M.,Kerwin,L.,andC.F.BarbasIII.(2003)Rapidmonoclonalantibodygenerationviadendriticcelltargetinginvivo.HybridomaandHybridomics22(1),23-31.

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