（原装进口）人尿激酶型纤溶酶原激活物受体（uPAR）Elisa试剂盒说明书

文档简介QuantikineHumanuPARImmunoassayCatalogNumberDUP00Forthequantitativedeterminationofhumanurokinase-typeplasminogenactivatorreceptor（uPAR）concentrationsincellculturesupernates,serum,plasma,andurine.Thispackageinsertmustbereadinitsentiretybeforeusingthisproduct.FORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.TABLEOFCONTENTSContentsPageINTRODUCTION2PRINCIPLEOFTHEASSAY..................................3LIMITATIONSOFTHEPROCEDURE3MATERIALSPROVIDED....................................3STORAGE4OTHERSUPPLIESREQUIRED.................................4PRECAUTION4SAMPLECOLLECTIONANDSTORAGE............................5SAMPLEPREPARATION5REAGENTPREPARATION...................................6ASSAYPROCEDURE7ASSAYPROCEDURESUMMARY...............................8CALCULATIONOFRESULTS9TYPICALDATA.........................................9TECHNICALHINTS10PRECISION...........................................10LINEARITY11SENSITIVITY..........................................11CALIBRATION11SAMPLEVALUES.......................................12SPECIFICITY13REFERENCES.........................................14PLATELAYOUT15MANUFACTUREDANDDISTRIBUTEDBY:R&DSystems,Inc.TELEPHONE:（800）343-7475614McKinleyPlaceNE（612）379-2956Minneapolis,MN55413FAX:（612）656-4400UnitedStatesofAmericaE-MAIL:info@RnDSystems.comDISTRIBUTEDBY:R&DSystemsEurope,Ltd.19BartonLaneTELEPHONE:+44（0）1235529449AbingdonScienceParkFAX:+44（0）1235533420Abingdon,OX143NBE-MAIL:info@RnDSystems.co.ukUnitedKingdomR&DSystemsChinaCo.Ltd.24A1HuaMinEmpirePlazaTELEPHONE:+86（21）52380373726WestYanAnRoadFAX:+86（21）52371001ShanghaiPRC200050E-MAIL:info@RnDSystemsChina.com.cnINTRODUCTIONUrokinase-typeplasminogenactivatorreceptor（uPAR,CD87）isacellsurfacereceptorthatbindsurokinase-typeplasminogenactivator（uPA）withhighaffinity,therebyfacilitatingthepericellularactivationofplasminogen（seereferences1and2forreviews）.uPAR,uPAandplasminogenactivatorinhibitor-1（PAI-1）,formatriadwithmultiplefunctions,includingregulationofcellattachment,migration,proliferationanddifferentiation,bybothproteolyticandnonproteolyticmechanisms（2）.uPARisanchoredtoextracellularsurfacesthroughaglycosylphosphatidylinositol（GPI）linkage,withnotransmembranedomain（3）.uPARissynthesizedasa335-amino-acidpolypeptidethatincludesa22-residuesignalpeptide（4）.A30-residuepeptideiscleavedfromtheC-terminusduringadditionoftheGPIanchor（3）.WithlossofthesignalpeptideandtheC-terminalpeptide,thematureproteinhas283aminoacids.Itisvariablyglycosylated,increasingitsmassfromabout31kDafortheproteinbackbonetoasmuchas55kDaforthematureglycoprotein（5）.Pro-uPA,asingle-chainprotein,isactivatedtoadisulfide-linked,two-chainproteinbyproteolyticcleavagebyplasmin（1,2,6）.Eitherpro-uPAortheactivetwo-chainuPAbindwithhighaffinitytouPAR.Thus,tracesofplasminactivatepro-uPAtouPA,leadingtoincreasinggenerationofplasmininapositivefeedbackloopthatisamplifiedbyuPAR.Whiletheinitiatingeventisnotclear,theeffectisthegenerationofplasminatthecellsurface,whereitdegradestheextracellularmatrixbyactivatingmatrixmetalloproteinases.Thisappearstobeakeyeventintumorinvasivenessandmetastasisandinmigrationofcellsingeneral（1,2,7）.ThefunctionsoftheuPA/uPARsystemare,however,moreextensivethanmediationofplasminformation,andtheyincludenon-proteolyticfunctions（1,2）.uPA/uPARislocalizedtofocalcontactpointsofcellswithinthesubstratum.Thesesitesincludeintracellularvinculinandtheextracellularadhesionmoleculevitronectin,suggestingdirectadhesivefunctionsandintracellularsignal领functionsforuPAR.uPARbindstointegrins,anditcontainschemotacticactivityinitssingleprotease-sensitiveregion.uPARhasbeenmeasuredinhumanplasma（7-9）.SolubleuPARisgeneratedbyremovaloftheGPIanchorbyanendogenousphospholipaseD,freeinguPARofitssurfaceattachment（10）.uPARiselevatedinplasmaofpatientswithparoxysmalnocturnalhemoglobinuria（7,8）,amanifestationofaninabilitytoaddGPIanchorstoproteins.IthasbeenpostulatedthattherealsomaybesolubleuPARduetoalternativesplicingoftheprimarytranscript（1）,ashasbeendemonstratedformouseuPAR（11）.uPARhasbeenidentifiedinurine,wherethelevelisaconsistentfractionofcreatinineconcentration（12）.TheQuantikineHumanuPARImmunoassayisa4.5hoursolid-phaseELISAdesignedtomeasurehumanuPARincellculturesupernates,serum,plasma,andurine.ItcontainsNS0-expressedrecombinanthumanuPARandantibodiesraisedagainsttherecombinantfactor.Ithasbeenshowntoaccuratelyquantitatetherecombinantfactor.ResultsobtainedusingnaturalhumanuPARshowedlinearcurvesthatwereparalleltothestandardcurvesobtainedusingtheQuantikinekitstandards.TheseresultsindicatethatthiskitcanbeusedtodeterminerelativemassvaluesofnaturalhumanuPAR.2PRINCIPLEOFTHEASSAYThisassayemploysthequantitativesandwichenzymeimmunoassaytechnique.AmonoclonalantibodyspecificforuPARhasbeenpre-coatedontoamicroplate.StandardsandsamplesarepipettedintothewellsandanyuPARpresentisboundbytheimmobilizedantibody.Afterwashingawayanyunboundsubstances,anenzyme-linkedpolyclonalantibodyspecificforuPARisaddedtothewells.Followingawashtoremoveanyunboundantibody-enzymereagent,asubstratesolutionisaddedtothewellsandcolordevelopsinproportiontotheamountofuPARboundintheinitialstep.Thecolordevelopmentisstoppedandtheintensityofthecolorismeasured.LIMITATIONSOFTHEPROCEDUREFORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.Thekit首ldnotbeusedbeyondtheexpirationdateonthekitlabel.Donotmixorsubstitutereagentswithotherlotsorsources.Ifsamplesfalloutsidethedynamicrangeoftheassay,furtherdilutethesampleswithCalibratorDiluentandrepeattheassay.Anyvariationinstandarddiluent,operator,pipettingtechnique,washingtechnique,incubationtimeortemperature,andkitagecancausevariationinbinding.Thisassayisdesignedtoeliminateinterferencebyligandsandotherproteinspresentinbiologicalsamples.UntilallfactorshavebeentestedintheQuantikineImmunoassay,thepossibilityofinterferencecannotbeexcluded.MATERIALSPROVIDEDuPARMicroplate（Part890714）-96wellpolystyrenemicroplate（12stripsof8wells）coatedwithamousemonoclonalantibodyagainstuPAR.uPARConjugate（Part890715）-21mLofpolyclonalantibodyagainstuPARconjugatedtohorseradishperoxidasewithpreservatives.uPARStandard（Part890716）-40ngofrecombinanthumanuPARinabufferedproteinbasewithpreservatives;lyophilized.AssayDiluentRD1W（Part895117）-11mLofabufferedproteinbasewithpreservatives.CalibratorDiluentRD6P（Part895118）-21mLofanimalserumwithpreservatives.WashBufferConcentrate（Part895003）-21mLofa25-foldconcentratedsolutionofbufferedsurfactantwithpreservatives.ColorReagentA（Part895000）-12.5mLofstabilizedhydrogenperoxide.ColorReagentB（Part895001）-12.5mLofstabilizedchromogen（tetramethylbenzidine）.StopSolution（Part895032）-6mLof2Nsulfuricacid.PlateCovers-4adhesivestrips.3STORAGEUnopenedKitStoreat2-8°C.Donotusepastkitexpirationdate.Opened/ReconstitutedReagentsDilutedWashBufferMaybestoredforupto1monthat2-8°C.*StopSolutionAssayDiluentRD1WCalibratorDiluentRD6PConjugateUnmixedColorReagentAUnmixedColorReagentBStandardMicroplateWellsReturnunusedwellstothefoilpouchcontainingthedesiccantpack,resealalongentireedgeofzip-seal.Maybestoredforupto1monthat2-8°C.**Providedthisiswithintheexpirationdateofthekit.OTHERSUPPLIESREQUIREDMicroplatereadercapableofmeasuringabsorbanceat450nm,withthecorrectionwavelengthsetat540nmor570nm.Pipettesandpipettetips.Deionizedordistilledwater.Squirtbottle,manifolddispenser,orautomatedmicroplatewasher.500mLgraduatedcylinder.Testtubesfordilution.HumanuPARControls（optional;availablefromR&DSystems）.PRECAUTIONTheStopSolutionprovidedwiththiskitisanacidsolution.Weareye,hand,face,andclothingprotectionwhenusingthismaterial.4SAMPLECOLLECTIONANDSTORAGECellCultureSupernates-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20°C.Avoidrepeatedfreeze-thawcycles.Serum-Useaserumseparatortube（SST）andallowsamplestoclotfor30minutesbeforecentrifugationfor10minutesat1000xg.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20°C.Avoidrepeatedfreeze-thawcycles.Plasma-CollectplasmausingheparinorEDTAasananticoagulant.Centrifugeat1000xgwithin30minutesofcollection.Assayimmediatelyoraliquotandstoresamplesat-20°C.Avoidrepeatedfreeze-thawcycles.Note:Citrateplasmahasnotbeenvalidatedforuseinthisassay.LipemicsamplesandsamplescontaininghighlevelsofhumanserumalbuminarenotsuitableforthemeasurementofhumanuPARwiththisassay.Urine-Asepticallycollectthefirsturineoftheday（mid-stream）,voideddirectlyintoasterilecontainer.Centrifugetoremoveparticulatematter.Assayimmediatelyoraliquotandstoreat-20°C.Avoidrepeatedfreeze-thawcycles.Formoreinformationoncollectingandhand领urinespecimens,refertotheNationalCommitteeforClinicalLaboratoryStandards（NCCLS）publicationGP16-T.SAMPLEPREPARATIONCellculturesupernates,serum,plasma,andurinesamplesrequireatleasta5-folddilution.Asuggested5-folddilutionis25Lofsample+100LofCalibratorDiluentRD6P.5REAGENTPREPARATIONBringallreagentstoroomtemperaturebeforeuse.WashBuffer-Ifcrystalshaveformedintheconcentrate,warmtoroomtemperatureandmixgentlyuntilthecrystalshavecompletelydissolved.Dilute20mLofWashBufferConcentrateintodeionizedordistilledwatertoprepare500mLofWashBuffer.SubstrateSolution-ColorReagentsAandB首ldbemixedtogetherinequalvolumeswithin15minutesofuse.Protectfromlight.200Loftheresultantmixtureisrequiredperwell.uPARStandard-ReconstitutetheuPARStandardwith1.0mLofdeionizedordistilledwater.Thisreconstitutionproducesastocksolutionof40,000pg/mL.Allowthestandardtositforaminimumof15minuteswithgentleagitationpriortomakingdilutions.Pipette450LofCalibratorDiluentRD6Pintothe4000pg/mLtube.Pipette200LofCalibratorDiluentRD6Pintotheremainingtubes.Usethestocksolutiontoproduceadilutionseries（below）.Mixeachtubethoroughlybeforethenexttransfer.The4000pg/mLstandardservesasthehighstandard.CalibratorDiluentRD6Pservesasthezerostandard（0pg/mL）.6ASSAYPROCEDUREBringallreagentsandsamplestoroomtemperaturebeforeuse.Itisrecommendedthatallsamples,standards,andcontrolsbeassayedinduplicate.1.Prepareallreagents,workingstandards,andsamplesasdirectedintheprevioussections.2.Removeexcessmicroplatestripsfromtheplateframe,returnthemtothefoilpouchcontainingthedesiccantpack,andreseal.3.Add100LofAssayDiluentRD1Wtoeachwell.4.Add50LofStandard,control,orsample*perwell.Coverwiththeadhesivestripprovided.Incubatefor2hoursatroomtemperature.Aplatelayoutisprovidedtorecordstandardsandsamplesassayed.5.Aspirateeachwellandwash,repeatingtheprocessthreetimesforatotaloffourwashes.Washbyfil领eachwellwithWashBuffer（400L）usingasquirtbottle,manifolddispenser,orautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.6.Add200LofuPARConjugatetoeachwell.Coverwithanewadhesivestrip.Incubatefor2hoursatroomtemperature.7.Repeattheaspiration/washasinstep5.8.Add200LofSubstrateSolutiontoeachwell.Incubatefor30minutesatroomtemperature.Protectfromlight.9.Add50LofStopSolutiontoeachwell.Thecolorinthewells首ldchangefrombluetoyellow.Ifthecolorinthewellsisgreenorifthecolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.10.Determinetheopticaldensityofeachwellwithin30minutes,usingamicroplatereadersetto450nm.Ifwavelengthcorrectionisavailable,setto540nmor570nm.Ifwavelengthcorrectionisnotavailable,subtractreadingsat540nmor570nmfromthereadingsat450nm.Thissubtractionwillcorrectforopticalimperfectionsintheplate.Readingsmadedirectlyat450nmwithoutcorrectionmaybehigherandlessaccurate.*Samplesrequiredilution.SeeSamplePreparationsection.7ASSAYPROCEDURESUMMARY8CALCULATIONOFRESULTSAveragetheduplicatereadingsforeachstandard,control,andsampleandsubtracttheaveragezerostandardopticaldensity.Createastandardcurvebyreducingthedatausingcomputersoftwarecapableofgeneratingalog/logcurve-fit.Asanalternative,constructastandardcurvebyplottingthemeanabsorbanceforeachstandardonthey-axisagainsttheconcentrationonthex-axisanddrawabestfitcurvethroughthepointsonalog/loggraph.ThedatamaybelinearizedbyplottingthelogoftheuPARconcentrationsversusthelogoftheO.D.onalinearscale,andthebestfitlinecanbedeterminedbyregressionanalysis.Ifthesampleshavebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.TYPICALDATAThisstandardcurveisprovidedfordemonstrationonly.Astandardcurve首ldbegeneratedforeachsetofsamplesassayed.9pg/mL062.5125250500100020004000O.D.0.0460.0440.0720.0730.1050.1050.1700.1710.3000.3040.5650.5451.0661.0752.1222.076Average0.0450.0720.1050.1700.3020.5551.0702.099Corrected___0.0270.0600.1250.2570.5101.0252.054TECHNICALHINTSWhenmixingorreconstitutingproteinsolutions,alwaysavoidfoaming.Toavoidcross-contamination,changepipettetipsbetweenadditionsofeachstandardlevel,betweensampleadditions,andbetweenreagentadditions.Also,useseparatereservoirsforeachreagent.Whenusinganautomatedplatewasher,addinga30secondsoakperiodfollowingtheadditionofwashbuffer,and/orrotatingtheplate180degreesbetweenwashstepsmayimproveassayprecision.Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.SubstrateSolution首ldremaincolorlessuntiladdedtotheplate.KeeptheSubstrateSolutionprotectedfromlight.SubstrateSolution首ldchangefromcolorlesstogradationsofblue.StopSolution首ldbeaddedtotheplateinthesameorderastheSubstrateSolution.ThecolordevelopedinthewellswillturnfrombluetoyellowuponadditionoftheStopSolution.WellsthataregreenincolorindicatethattheStopSolutionhasnotmixedthoroughlywiththeSubstrateSolution.PRECISIONIntra-assayPrecision（Precisionwithinanassay）Threesamplesofknownconcentrationweretestedtwentytimesononeplatetoassessintra-assayprecision.Inter-assayPrecision（Precisionbetweenassays）Threesamplesofknownconcentrationweretestedinfortyseparateassaystoassessinter-assayprecision.Intra-assayPrecisionInter-assayPrecisionSample123123n202020404040Mean（pg/mL）8361593241279615462300Standarddeviation17.365.418144.378.9136CV（%）2.14.17.55.65.15.910LINEARITYToassessthelinearityoftheassay,samplesspikedwithhighconcentrationsofuPARweredilutedwithCalibratorDiluentRD6Ptoproducesampleswithvalueswithinthedynamicrangeoftheassay.Cellculturemedia*（n=4）Serum*（n=5）Heparinplasma*（n=5）EDTAplasma*（n=5）Urine*（n=5）1:2Average%ofExpected10396-112104101-105104101-10610194-105102Range（%）99-1051:4Average%ofExpected109101-115106103-111107104-110105101-108104Range（%）95-1121:8Average%ofExpected109101-111106103-112106102-110106103-113108Range（%）104-1131:16Average%ofExpected10999-11410599-11110598-11010498-111108Range（%）103-112*Sampleswerediluted5-foldpriortoassayasdirectedinSamplePreparation.SENSITIVITYTheminimumdetectabledose（MDD）ofuPARistypicallylessthan33pg/mL.TheMDDwasdeterminedbyaddingtwostandarddeviationstothemeanopticaldensityvalueoftwentyzerostandardreplicatesandcalculatingthecorrespondingconcentration.CALIBRATIONThisimmunoassayiscalibratedagainstahighlypurifiedNS0-expressedrecombinanthumanuPARproducedatR&DSystems.11SAMPLEVALUESSerum/Plasma/Urine-SamplesfromapparentlyhealthyvolunteerswereevaluatedforthepresenceofuPARinthisassay.Nomedicalhistorieswereavailableforthedonorsusedinthisstudy.SampleTypeMean（pg/mL）Range（pg/mL）Serum（n=60）23701195-4415Heparinplasma（n=35）2165977-5347EDTAplasma（n=35）1871864-3829Urine（n=35）2975691-6098CellCultureSupernates-Humanperipheralbloodmononuclearcells（5x106cells/mL）wereculturedinRPMIsupplementedwith5%fetalcalfserum,50M-mercaptoethanol,2mML-glutamine,100U/mLpenicillin,and100g/mLstreptomycinsulfate.Thecellswereculturedunstimulatedorstimulatedwith10g/mLPHA.AliquotsofthecellculturesupernateswereremovedandassayedforlevelsofnaturaluPAR.ConditionDay1（pg/mL）Day5（pg/mL）Unstimulated12282123Stimulated10,005689512SPECIFICITYThisassayrecognizesrecombinantandnaturalhumanuPAR.Thefactorslistedbelowwerepreparedat50ng/mLinCalibratorDiluentRD6Pandassayedforcross-reactivity.Preparationsofthefollowingfactorsat50ng/mLinamid-rangerecombinanthumanuPARcontrolwereassayedforinterference.Nosignificantcross-reactivityorinterferencewasobserved.RecombinanthumanuPAdoesnotcross-reactinthisassaybutdoesinterfereatconcentrationsgreaterthan3000pg/mL.13Recombinanthuman:ANGARCNTF-ECGFEGFEpoFGFacidicFGFbasicFGF-4FGF-5FGF-6Flt-3LigandG-CSFGM-CSFsgp130GROGROGROHB-EGFHGFIFN-IGF-IIGF-IIIL-1IL-1IL-1raIL-1sRIIL-1sRIIIL-2IL-2sRIL-3IL-3sRIL-4IL-4sRIL-5IL-5sRIL-5sRIL-6IL-6sRIL-7IL-8IL-9IL-10IL-11IL-12IL-13KGF（FGF-7）LAP（TGF-1）LIFM-CSFMCP-1MIP-1MIP-1-NGFOSMPD-ECGFPDGF-AAPDGF-ABPDGF-BBPTNRANTESSCFSLPITGF-TGF-1TGF-2TGF-3TGF-sRIITNF-TNF-sTNFRIsTNFRIIVEGFRecombinantmouse:GM-CSFIL-1IL-1IL-3IL-4IL-5IL-6IL-7IL-9IL-10IL-13LIFMIP-1MIP-1SCFTNF-uPARRecombinantamphibian:TGF-5Naturalproteins:bovineFGFacidicbovineFGFbasichumanPDGFporcinePDGFhumanTGF-1porcineTGF-1REFERENCES1.Dear,A.E.andR.L.Medcalf（1998）Eur.J.Biochem.252:185.2.Blasi,F.（1997）ImmunlogyToday18:415.3.Ploug,M.etal.（1991）J.Biol.Chem.266:1926.4.Roldan,A.L.（1990）EMBOJ.9:467.5.Behrendt,N.（1990）J.Biol.Chem.265:6453.6.Blasi,F.etal.（1987）J.CellBiol.104:801.7.Ronne,E.etal.（1995）Br.J.Haematol.89:576.8.Ploug,M.etal.（1992）Blood79:1447.9.Stephens,R.W.etal.（1999）J.Natl.CancerInst.91:869.10.Wilhelm,O.G.etal.（1999）J.CellularPhysiol.180:225.11.Kristensen,P.etal.（1991）J.CellBiol.115:1763.12.Sier,C.F.M.etal.（1999）Lab.Invest.79:717.14PLATELAYOUTUsethisplatelayouttorecordstandardsandsamplesassayed.2010R&DSystems,Inc.12.99750440.57/10