Human VEGF Immunoassay Quantikine ELISA

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QuantikineELISAThispackageinsertmustbereadinitsentiretybeforeusingthisproduct.Forresearchuseonly.Notforuseindiagnosticprocedures.CatalogNumberDVE00CatalogNumberSVE00CatalogNumberPDVE00ForthequantitativedeterminationofhumanVascularEndothelialGrowthFactor（VEGF）concentrationsincellculturesupernates,serum,andplasma.MANUFACTUREDANDDISTRIBUTEDBY:USA&Canada|R&DSystems,Inc.614McKinleyPlaceNE,Minneapolis,MN55413,USATEL:（800）343-7475（612）379-2956FAX:（612）656-4400E-MAIL:info@RnDSystems.comDISTRIBUTEDBY:UK&Europe|R&DSystemsEurope,Ltd.19BartonLane,AbingdonSciencePark,AbingdonOX143NB,UKTEL:+44（0）1235529449FAX:+44（0）1235533420E-MAIL:info@RnDSystems.co.ukChina|R&DSystemsChinaCo.,Ltd.24A1HuaMinEmpirePlaza,726WestYanAnRoad,ShanghaiPRC200050TEL:+86（21）52380373FAX:+86（21）52371001E-MAIL:info@RnDSystemsChina.com.cnTABLEOFCONTENTSSECTIONPAGEINTRODUCTION.....................................................................................................................................................................1PRINCIPLEOFTHEASSAY...................................................................................................................................................2LIMITATIONSOFTHEPROCEDURE.................................................................................................................................2TECHNICALHINTS.................................................................................................................................................................2MATERIALSPROVIDED&STORAGECONDITIONS...................................................................................................3OTHERSUPPLIESREQUIRED.............................................................................................................................................4PRECAUTIONS.........................................................................................................................................................................4SAMPLECOLLECTION&STORAGE.................................................................................................................................4REAGENTPREPARATION.....................................................................................................................................................SSAYPROCEDURE.............................................................................................................................................................6CALCULATIONOFRESULTS...............................................................................................................................................7TYPICALDATA.........................................................................................................................................................................7PRECISION................................................................................................................................................................................8RECOVERY................................................................................................................................................................................8SENSITIVITY.............................................................................................................................................................................8LINEARITY.................................................................................................................................................................................9CALIBRATION..........................................................................................................................................................................9SAMPLEVALUES..................................................................................................................................................................10SPECIFICITY...........................................................................................................................................................................11REFERENCES.........................................................................................................................................................................12PLATELAYOUT.....................................................................................................................................................................13www.RnDSystems.com1INTRODUCTIONVascularendothelialgrowthfactor（VEGForVEGF-A）,alsoknownasvascularpermeabilityfactor（VPF）,isapotentmediatorofbothangiogenesisandvasculogenesisinthefetusandadult（1-3）.ItisamemberofthePDGFfamilythatischaracterizedbythepresenceofeightconservedcysteineresiduesinacystineknotstructureandtheformationofantiparalleldisulfide-linkeddimers（4）.Humansexpressalternatelysplicedisoformsof121,145,165,183,189,and206aminoacids（aa）inlength（4）.VEGF165appearstobethemostabundantandpotentisoform,followedbyVEGF121andVEGF189（3,4）.IsoformsotherthanVEGF121containbasicheparin-bindingregionsandarenotfreelydiffusible（4）.HumanVEGF165shares88%aasequenceidentitywithcorrespondingregionsofmouseandratVEGF.VEGFisexpressedinmultiplecellsandtissuesincludingskeletalandcardiacmuscle（5,6）,hepatocytes（7）,osteoblasts（8）,neutrophils（9）,macrophages（10）,keratinocytes（11）,brownadiposetissue（12）,CD34+stemcells（13）,endothelialcells（14）,fibroblasts,andvascularsmoothmusclecells（15）.VEGFexpressionisinducedbyhypoxiaandcytokinessuchasIL-1,IL-6,IL-8,oncostatinMandTNF-α（3,4,9,16）.VEGFisoformsaredifferentiallyexpressedduringdevelopmentandintheadult（3）.VEGFdimersbindtotworelatedreceptortyrosinekinases,VEGFR1（alsocalledFlt-1）andVEGFR2（Flk-1/KDR）,andinducetheirhomodimerizationandautophosphorylation（3,4,7,17,18）.Thesereceptorshavesevenextracellularimmunoglobulin-likedomainsandanintracellularsplittyrosinekinasedomain.Theyareexpressedonvascularendothelialcellsandarangeofnon-endothelialcells.AlthoughVEGFaffinityishighestforbindingtoVEGFR1,VEGFR2appearstobetheprimarymediatorofVEGFangiogenicactivity（3,4）.VEGF165alsobindsthesemaphorinreceptor,neuropilin-1,whichpromotescomplexformationwithVEGFR2（19）.VEGFisbestknownforitsroleinvasculogenesis.Duringembryogenesis,VEGFregulatestheproliferation,migration,andsurvivalofendothelialcells（3,4）,thusregulatingbloodvesseldensityandsize,butplayingnoroleindeterminingvascularpatterns.VEGFpromotesboneformationthroughosteoblastandchondroblastrecruitmentandisalsoamonocytechemoattractant（20-22）.Afterbirth,VEGFmaintainsendothelialcellintegrityandisapotentmitogenformicro-andmacro-vascularendothelialcells.Inadults,VEGFfunctionsmainlyinwoundhea领andthefemalereproductivecycle（3）.Indiseasedtissues,VEGFpromotesvascularpermeability.Itisthusthoughttocontributetotumormetastasisbypromotingbothextravasationandtumorangiogenesis（23,24）.VariousstrategieshavebeenemployedtherapeuticallytoantagonizeVEGF-mediatedtumorangiogenesis（25）.CirculatingVEGFlevelscorrelatewithdiseaseactivityinautoimmunediseasessuchasrheumatoidarthritis,multiplesclerosisandsystemiclupuserythematosus（26）.TheQuantikineHumanVEGFImmunoassayisa4.5hoursolidphaseELISAdesignedtomeasureVEGF165levelsincellculturesupernates,serum,andplasma.ItcontainsSf21-expressedrecombinanthumanVEGF165andantibodiesraisedagainsttherecombinantprotein.ResultsobtainedfornaturallyoccurringhumanVEGFandrecombinanthumanVEGF121showedlinearcurvesthatwereparalleltothestandardcurvesobtainedusingtheQuantikineHumanVEGFImmunoassaystandards.TheseresultsindicatethatthiskitcanbeusedtodeterminerelativemassvaluesfornaturalhumanVEGF.2Forresearchuseonly.Notforuseindiagnosticprocedures.PRINCIPLEOFTHEASSAYThisassayemploysthequantitativesandwichenzymeimmunoassaytechnique.AmonoclonalantibodyspecificforVEGFhasbeenpre-coatedontoamicroplate.StandardsandsamplesarepipettedintothewellsandanyVEGFpresentisboundbytheimmobilizedantibody.Afterwashingawayanyunboundsubstances,anenzyme-linkedpolyclonalantibodyspecificforVEGFisaddedtothewells.Followingawashtoremoveanyunboundantibody-enzymereagent,asubstratesolutionisaddedtothewellsandcolordevelopsinproportiontotheamountofVEGFboundintheinitialstep.Thecolordevelopmentisstoppedandtheintensityofthecolorismeasured.LIMITATIONSOFTHEPROCEDUREFORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.Thekit首ldnotbeusedbeyondtheexpirationdateonthekitlabel.Donotmixorsubstitutereagentswiththosefromotherlotsorsources.ItisimportantthattheCalibratorDiluentselectedforthestandardcurvebeconsistentwiththesamplesbeingassayed.Ifsamplesgeneratevalueshigherthanthehigheststandard,dilutethesampleswiththeappropriateCalibratorDiluentandrepeattheassay.Anyvariationinstandarddiluent,operator,pipettingtechnique,washingtechnique,incubationtimeortemperature,andkitagecancausevariationinbinding.Variationsinsamplecollection,processing,andstoragemaycausesamplevaluedifferences.Thisassayisdesignedtoeliminateinterferencebysolublereceptors,bindingproteins,andotherfactorspresentinbiologicalsamples.UntilallfactorshavebeentestedintheQuantikineImmunoassay,thepossibilityofinterferencecannotbeexcluded.TECHNICALHINTSWhenmixingorreconstitutingproteinsolutions,alwaysavoidfoaming.Toavoidcross-contamination,changepipettetipsbetweenadditionsofeachstandardlevel,betweensampleadditions,andbetweenreagentadditions.Also,useseparatereservoirsforeachreagent.Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.Whenusinganautomatedplatewasher,addinga30secondsoakperiodfollowingtheadditionofWashBuffer,and/orrotatingtheplate180degreesbetweenwashstepsmayimproveassayprecision.SubstrateSolution首ldremaincolorlessuntiladdedtotheplate.KeepSubstrateSolutionprotectedfromlight.SubstrateSolution首ldchangefromcolorlesstogradationsofblue.StopSolution首ldbeaddedtotheplateinthesameorderastheSubstrateSolution.ThecolordevelopedinthewellswillturnfrombluetoyellowuponadditionoftheStopSolution.WellsthataregreenincolorindicatethattheStopSolutionhasnotmixedthoroughlywiththeSubstrateSolution.www.RnDSystems.com3MATERIALSPROVIDED&STORAGECONDITIONSStoretheunopenedkitat2-8°C.Donotusepastkitexpirationdate.PARTPART#CATALOG#DVE00CATALOG#SVE00DESCRIPTIONSTORAGEOFOPENED/RECONSTITUTEDMATERIALVEGFMicroplate8902181plate6plates96wellpolystyrenemicroplate（12stripsof8wells）coatedwithamousemonoclonalantibodyagainstVEGF.Returnunusedwellstothefoilpouchcontainingthedesiccantpack.Resealalongentireedgeofzip-seal.Maybestoredforupto1monthat2-8°C.*VEGFStandard8902203vials18vials2000pg/vialofrecombinantVEGF165inabufferedproteinbasewithpreservatives;lyophilized.DiscardtheVEGFstocksolutionanddilutionsafter4hours.Useafreshstandardforeachassay.VEGFConjugate8902191vial6vials21mL/vialofapolyclonalantibodyagainstVEGFconjugatedtohorseradishperoxidasewithpreservatives.Maybestoredforupto1monthat2-8°C.*AssayDiluentRD1W8951171vial6vials11mL/vialofabufferedproteinbasewithpreservatives.CalibratorDiluentRD5K8951191vial6vials21mL/vialofabufferedproteinbasewithpreservatives.Forcellculturesupernatesamples.CalibratorDiluentRD6U8951481vial6vials21mL/vialofanimalserumwithpreservatives.Forserum/plasmasamples.WashBufferConcentrate8950031vial6vials21mL/vialofa25-foldconcentratedsolutionofbufferedsurfactantwithpreservatives.ColorReagentA8950001vial6vials12mL/vialofstabilizedhydrogenperoxide.ColorReagentB8950011vial6vials12mL/vialofstabilizedchromogen（tetramethylbenzidine）.StopSolution8950321vial6vials6mL/vialof2Nsulfuricacid.PlateSealersN/A4strips24stripsAdhesivestrips.*Providedthisiswithintheexpirationdateofthekit.DVE00containssufficientmaterialstorunanELISAonone96wellplate.SVE00（SixPak）containssufficientmaterialstorunELISAsonsix96wellplates.ThiskitisalsoavailableinaPharmPak（R&DSystems,Catalog#PDVE00）.PharmPakscontainsufficientmaterialstorunELISAson50microplates.Specificvialcountsofeachcomponentmayvary.Pleaserefertotheliteratureaccompanyingyourorderforspecificvialcounts.4Forresearchuseonly.Notforuseindiagnosticprocedures.OTHERSUPPLIESREQUIREDMicroplatereadercapableofmeasuringabsorbanceat450nm,withthecorrectionwavelengthsetat540nmor570nm.Pipettesandpipettetips.Deionizedordistilledwater.Squirtbottle,manifolddispenser,orautomatedmicroplatewasher.500mLgraduatedcylinder.Polypropylenetesttubesfordilutionofstandards.HumanVEGFControls（optional;availablefromR&DSystems）.PRECAUTIONSCalibratorDiluentRD6Ucontainssodiumazidewhichmayreactwithleadandcopperplumbingtoformexplosivemetallicazides.Flushwithlargevolumesofwaterduringdisposal.VEGFisdetectableinsaliva.Takeprecautionarymeasurestopreventcontaminationofthekitreagentswhilerunningtheassay.TheStopSolutionprovidedwiththiskitisanacidsolution.Wearprotectivegloves,clothing,eye,andfaceprotection.Washhandsthoroughlyafterhand领.SAMPLECOLLECTION&STORAGEThesamplecollectionandstorageconditionslistedbelowareintendedasgeneralguidelines.Samplestabilityhasnotbeenevaluated.CellCultureSupernates-Cellculturesupernates首ldcontainatleast1%fetalcalfserumforstabilityoftheVEGF.Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat≤-20°C.Avoidrepeatedfreeze-thawcycles.Serum-Useaserumseparatortube（SST）andallowsamplestoclotfor30minutesbeforecentrifugationfor15minutesat1000xg.Removeserumandassayimmediatelyoraliquotandstoresamplesat≤-20°C.Avoidrepeatedfreeze-thawcycles.Plasma-CollectplasmausingEDTA,heparin,orcitrateasananticoagulant.Centrifugefor15minutesat1000xgwithin30minutesofcollection.Assayimmediatelyoraliquotandstoresamplesat≤-20°C.Avoidrepeatedfreeze-thawcycles.www.RnDSystems.com5REAGENTPREPARATIONBringallreagentstoroomtemperaturebeforeuse.WashBuffer-Ifcrystalshaveformedintheconcentrate,warmtoroomtemperatureandmixgentlyuntilthecrystalshavecompletelydissolved.Dilute20mLofWashBufferConcentrateintodeionizedordistilledwatertoprepare500mLofWashBuffer.SubstrateSolution-ColorReagentsAandB首ldbemixedtogetherinequalvolumeswithin15minutesofuse.Protectfromlight.200μLoftheresultantmixtureisrequiredperwell.VEGFStandard-ReconstitutetheVEGFStandardwith1.0mLofCalibratorDiluentRD5K（forcellculturesupernatesamples）orCalibratorDiluentRD6U（forserum/plasmasamples）.Thisreconstitutionproducesastocksolutionof2000pg/mL.Allowthestandardtositforaminimumof15minuteswithgentleagitationpriortomakingdilutions.ForCellCultureSupernateSamples:Usepolypropylenetubes.Pipette500μLofCalibratorDiluentRD5Kintoeachtube.Usethestocksolutiontoproduceadilutionseries（below）.Mixeachtubethoroughlybeforethenexttransfer.The1000pg/mLdilutionservesasthehighstandard.CalibratorDiluentRD5Kservesasthezerostandard（0pg/mL）.500μLStd.2000pg/mL1000pg/mL500pg/mL250pg/mL125pg/mL62.5pg/mL31.2pg/mL15.6pg/mL500μL500μL500μL500μL500μL500μL500μLStd.2000pg/mL1000pg/mL500pg/mL250pg/mL125pg/mL62.5pg/mL31.2pg/mL500μL500μL500μL500μL500μLForSerum/PlasmaSamples:Usepolypropylenetubes.Pipette500μLofCalibratorDiluentRD6Uintoeachtube.Usethestocksolutiontoproduceadilutionseries（below）.Mixeachtubethoroughlybeforethenexttransfer.Theundilutedstandardservesasthehighstandard（2000pg/mL）.CalibratorDiluentRD6Uservesasthezerostandard（0pg/mL）6Forresearchuseonly.Notforuseindiagnosticprocedures.ASSAYPROCEDUREBringallreagentsandsamplestoroomtemperaturebeforeuse.Itisrecommendedthatallstandards,samples,andcontrolsbeassayedinduplicate.1.Prepareallreagents,workingstandards,andsamplesasdirectedintheprevioussections.2.Removeexcessmicroplatestripsfromtheplateframe,returnthemtothefoilpouchcontainingthedesiccantpack,andreseal.3.ForCellCultureSupernateSamples:Add50μLofAssayDiluentRD1Wtoeachwell.ForSerum/PlasmaSamples:Add100μLofAssayDiluentRD1Wtoeachwell.4.ForCellCultureSupernateSamples:Add200μLofStandard,control,orsampleperwell.ForSerum/PlasmaSamples:Add100μLofStandard,control,orsampleperwell.Coverwiththeadhesivestripprovidedandincubatefor2hoursatroomtemperature.Aplatelayoutisprovidedtorecordthestandardsandsamplesassayed.5.Aspirateeachwellandwash,repeatingtheprocesstwiceforatotalofthreewashes.Washbyfil领eachwellwithWashBuffer（400μL）usingasquirtbottle,manifolddispenser,orautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.6.Add200μLofVEGFConjugatetoeachwell.Coverwithanewadhesivestrip.Incubatefor2hoursatroomtemperature.7.Repeattheaspiration/washasinstep5.8.Add200μLofSubstrateSolutiontoeachwell.Protectfromlight.ForCellCultureSupernateSamples:Incubatefor20minutesatroomtemperature.ForSerum/PlasmaSamples:Incubatefor25minutesatroomtemperature.9.Add50μLofStopSolutiontoeachwell.Ifcolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.Ifthecolorinthewellsisgreenorthecolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.10.Determinetheopticaldensityofeachwellwithin30minutes,usingamicroplatereadersetto450nm.Ifwavelengthcorrectionisavailable,setto540nmor570nm.Ifwavelengthcorrectionisnotavailable,subtractreadingsat540nmor570nmfromthereadingsat450nm.Thissubtractionwillcorrectforopticalimperfectionsintheplate.Readingsmadedirectlyat450nmwithoutcorrectionmaybehigherandlessaccurate.www.RnDSystems.com7CALCULATIONOFRESULTSAveragetheduplicatereadingsforeachstandard,control,andsampleandsubtracttheaveragezerostandardopticaldensity.Createastandardcurvebyreducingthedatausingcomputersoftwarecapableofgeneratingafourparameterlogistic（4-PL）curvefit.Asanalternative,constructastandardcurvebyplottingthemeanabsorbanceforeachstandardonthey-axisagainsttheconcentrationonthex-axisanddrawabestfitcurvethroughthepointsonthegraph.ThedatamaybelinearizedbyplottingthelogoftheVEGFconcentrationsversusthelogoftheO.D.andthebestfitlinecanbedeterminedbyregressionanalysis.Thisprocedurewillproduceanadequatebutlessprecisefitofthedata.Ifsampleshavebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.TYPICALDATAThesestandardcurvesareprovidedfordemonstrationonly.Astandardcurve首ldbegeneratedforeachsetofsamplesassayed.（pg/mL）O.D.AverageCorrected00.0740.0750.07615.60.1180.1200.0450.12131.20.1590.1590.0840.15962.50.2460.2440.1690.2421250.3840.3810.3060.3782500.6660.6680.5930.6695001.2581.2601.1851.26310002.3022.2682.1932.233（pg/mL）O.D.AverageCorrected00.0680.0700.07131.20.1070.1080.0380.11062.50.1490.1510.0810.1531250.2300.2300.1600.2302500.3770.3820.3120.3875000.6570.6780.6080.69910001.2611.2711.2011.28120002.1592.2022.1322.246CALIBRATORDILUENTRD5KCALIBRATORDILUENTRD6U8Forresearchuseonly.Notforuseindiagnosticprocedures.PRECISIONIntra-assayPrecision（Precisionwithinanassay）Threesamplesofknownconcentrationweretestedtwentytimesononeplatetoassessintraassayprecision.Inter-assayPrecision（Precisionbetweenassays）Threesamplesofknownconcentrationweretestedinfortyseparateassaystoassessinterassayprecision.CELLCULTURESUPERNATEASSAYIntra-AssayPrecisionInter-AssayPrecisionSample123123n202020404040Mean（pg/mL）29.112353132.8128495Standarddeviation1.95.018.42.86.433.0CV（%）6.54.13.58.55.06.7SERUM/PLASMAASSAYIntra-AssayPrecisionInter-AssayPrecisionSample123123n202020404040Mean（pg/mL）53.723591064.52501003Standarddeviation3.610.646.25.717.461.7CV（%）6.74.55.18.87.06.2RECOVERYTherecoveryofVEGFspikedtothreedifferentlevelsthroughouttherangeoftheassayinvariousmatriceswasevaluated.SampleTypeAverage%RecoveryRangeCellculturemedia（n=5）10295-111%Serum（n=5）10292-115%EDTAplasma（n=5）9782-113%Heparinplasma（n=5）9382-102%Citrateplasma（n=5）10088-113%SENSITIVITYUsingCalibratorDiluentRD5Ktheminimumdetectabledose（MDD）ofVEGFistypicallylessthan5.0pg/mL.UsingCalibratorDiluentRD6UtheMDDistypicallylessthan9.0pg/mL.TheMDDwasdeterminedbyaddingtwostandarddeviationstothemeanopticaldensityvalueoftwentyzerostandardreplicatesandcalculatingthecorrespondingconcentration.www.RnDSystems.com9LINEARITYToassesslinearityoftheassay,sampleswerespikedwithhighconcentrationsofVEGFanddilutedwiththeappropriateCalibratorDiluenttoproducesampleswithvalueswithinthedynamicrangeoftheassay.Cellculturemedia（n=5）Serum（n=5）EDTAplasma（n=5）Heparinplasma（n=5）Citrateplasma（n=5）1:2Average%ofExpected9897979495Range（%）94-10091-10382-10787-9990-1001:4Average%ofExpected9697989394Range（%）93-9993-10491-10685-9889-991:8Average%ofExpected9396969292Range（%）88-10293-10389-10685-10185-971:16Average%ofExpected9394949492Range（%）88-10591-10184-10683-10385-98CALIBRATIONThisimmunoassayiscalibratedagainstahighlypurifiedSf21-expressedrecombinanthumanVEGF165producedatR&DSystems.TheNIBSC/WHOVEGF165preparation02/286（recombinanthumanDNA）wasevaluatedinthiskit.Thedoseresponsecurveofthestandard02/286parallelstheQuantikinestandardcurve.ToconvertsamplevaluesobtainedwiththeQuantikineHumanVEGFkittoapproximateNIBSC/WHO02/286Units,usetheequationbelow.NIBSC/WHO（02/286）approximatevalue（U/mL）=0.002xQuantikineVEGFvalue（pg/mL）Note:BasedondatageneratedinApril2011.10Forresearchuseonly.Notforuseindiagnosticprocedures.SAMPLEVALUESSerum/Plasma-SamplesfromapparentlyhealthyvolunteerswereevaluatedforthepresenceofVEGFinthisassay.Nomedicalhistorieswereavailableforthedonorsusedinthisstudy.SampleTypeMeanofDetectable（pg/mL）%DetectableRange（pg/mL）Serum（n=37）22010062-707EDTAplasma（n=37）6124ND-115Heparinplasma（n=37）4122ND-55Citrateplasma（n=37）___0NDND=Non-detectableCellCultureSupernates-Humanperipheralbloodmononuclearcells（1x106cells/mL）wereculturedinRPMIsupplementedwith5%fetalcalfserum,50μMβ-mercaptoethanol,2mML-glutamine,100U/mLpenicillin,and100μg/mLstreptomycinsulfate.Thecellswereculturedunstimulatedorstimulatedwith10μg/mLPHAfor1and5days.AliquotsofthecellculturesupernateswereremovedandassayedforlevelsofnaturalVEGF.ConditionDay1（pg/mL）Day5（pg/mL）Unstimulated356332Stimulated141440www.RnDSystems.com11SPECIFICITYThisassayrecognizesnaturalandrecombinanthumanVEGF.ThisassayalsorecognizesrecombinanthumanVEGF165b.Thefactorslistedbelowwerepreparedat50ng/mLinCalibratorDiluentandassayedforcrossreactivity.Preparationsofthefollowingfactorsat50ng/mLinamid-rangeVEGFcontrolwereassayedforinterference.Thefollowingfactorsshowednocross-reactivityorinterference.Recombinanthuman:PDGF-AAPDGF-ABPDGF-BBPDGF-CCPDGF-DDPlGFPlGF-2VEGF165/PlGFVEGF-B167VEGF-CVEGF-DVEGFR3Recombinantmouse:PDGF-CCPlGF-2VEGF120VEGF164VEGFR3Recombinantrat:PDGF-AAPDGF-ABPDGF-BBVEGF164Recombinantzebrafish:VEGFNaturalproteins:humanPDGFporcinePDGFVEGF-relatedfactorsshowingcross-reactivityorinterference.RecombinanthumanVEGFR1/Flt-1Interferenceatlevels≥500pg/mLRecombinanthumanVEGFR2/KDRInterferenceatlevels≥2000pg/mLRecombinantmouseVEGFR1/Flk-1Interferenceatlevels≥500pg/mLRecombinantmouseVEGFR2/KDRInterferenceatlevels≥4000pg/mLRecombinantcanineVEGFCross-reactsapproximately67%RecombinantfelineVEGFCross-reactsapproximately82%12Forresearchuseonly.Notforuseindiagnosticprocedures.REFERENCES1.Leung,D.W.etal.（1989）Science246:1306.2.Keck,P.J.etal.（1989）Science246:1309.3.Byrne,A.M.etal.（2005）J.Cell.Mol.Med.9:777.4.Robinson,C.J.andS.E.Stringer（2001）J.Cell.Sci.114:853.5.Richardson,R.S.etal.（1999）Am.J.Physiol.277:H2247.6.Sugishita,Y.etal.（2000）Biochem.Biophys.Res.Commun.268:657.7.Yamane,A.etal.（1994）Oncogene9:2683.8.Goad,D.L.etal.（1996）Endocrinology137:2262.9.Gaudry,M.etal.（1997）Blood90:4153.10.Mclaren,J.etal.（1996）J.Clin.Invest.98:482.11.Diaz,B.V.etal.（2000）J.Biol.Chem.275:642.12.Asano,A.etal.（1997）Biochem.J.328:179.13.Bautz,F.etal.（2000）Exp.Hematol.28:700.14.Namiki,A.etal.（1995）J.Biol.Chem.270:31189.15.Nauck,M.etal.（1997）Am.J.Respir.Cell.Mol.Biol.16:398.16.Angelo,L.S.andR.Kurzrock（2007）Clin.CancerRes.13:2825.17.Neufeld,G.etal.（1999）FASEB.J.13:9.18.Kowalewski,M.P.etal.（2005）Accession#ABB82619.19.Pan,Q.etal.（2007）J.Biol.Chem.282:24049.20.Dai,J.andA.B.Rabie（2007）J.Dent.Res.86:937.21.Breier,G.（2000）Semin.Thromb.Hemost.26:553.22.Barleon,B.etal.（1996）Blood87:3336.23.Weis,S.M.andD.A.Cheresh（2005）Nature437:497.24.Thurston,G.（2002）J.Anat.200:575.25.Grothey,A.andE.Galanis（2009）Nat.Rev.Clin.Oncol.6:507.26.Carvalho,J.F.etal.（2007）J.Clin.Immunol.27:246.www.RnDSystems.com13PLATELAYOUTUsethisplatelayouttorecordstandardsandsamplesassayed.14Forresearchuseonly.Notforuseindiagnosticprocedures.
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