检测TNF的 elisa试剂盒说明书.docx
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检测TNF的 elisa试剂盒说明书.docx
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检测TNF的elisa试剂盒说明书
QuantikineÒ
RatTNF-aImmunoassay
CatalogNumberRTA00
SRTA00
PRTA00
Forthequantitativedeterminationofrattumornecrosisfactoralpha(TNF-aconcentrationsincellculturesupernates,ratserum,andplasma.
Thispackageinsertmustbereadinitsentiretybeforeusingthisproduct.
FORRESEARCHUSEONLY.
NOTFORUSEINDIAGNOSTICPROCEDURES.
TABLEOFCONTENTS
ContentsPage
INTRODUCTION2PRINCIPLEOFTHEASSAY..................................3LIMITATIONSOFTHEPROCEDURE3PRECAUTION..........................................3TECHNICALHINTS3MATERIALSPROVIDED....................................4STORAGE5OTHERSUPPLIESREQUIRED.................................5SAMPLECOLLECTIONANDSTORAGE6SAMPLEPREPARATION....................................6REAGENTPREPARATION7ASSAYPROCEDURE......................................8PROCEDURESUMMARYANDCHECKLIST9CALCULATIONOFRESULTS.................................10TYPICALDATA10PRECISION..........................................11RECOVERY11LINEARITY...........................................12SENSITIVITY12CALIBRATION.........................................13SAMPLEVALUES13SPECIFICITY.........................................13REFERENCES14PLATELAYOUT........................................15
MANUFACTUREDANDDISTRIBUTEDBY:
R&DSystems,Inc.TELEPHONE:
(800343-7475
614McKinleyPlaceNE(612379-2956
Minneapolis,MN55413FAX:
(612656-4400
UnitedStatesofAmericaE-MAIL:
info@RnDS
DISTRIBUTEDBY:
R&DSystemsEurope,Ltd.
19BartonLaneTELEPHONE:
+44(01235529449
AbingdonScienceParkFAX:
+44(01235533420
Abingdon,OX143NBE-MAIL:
info@RnDSystems.co.uk
UnitedKingdom
R&DSystemsChinaCo.Ltd.
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+86(2152380373
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+86(2152371001
ShanghaiPRC200050E-MAIL:
info@RnDSystemsC
INTRODUCTION
Tumornecrosisfactoralpha(TNF-a,alsoknownascachectin;andtumornecrosisfactorbeta(TNF-b,alsoknownaslymphotoxin,aretwocloselyrelatedproteins(approximately34%aminoacidsequenceidentitythatbindtothesamecellsurfacereceptorsandshowmanycommonbiologicalfunctions.TNF-aand-bplaycriticalrolesinnormalhostresistancetoinfectionandtothegrowthofmalignanttumors,servingasimmunostimulantsandasmediatorsoftheinflammatoryresponse.Over-productionofTNFs,however,hasbeenimplicatedasplayingaroleinanumberofpathologicalconditions,includingcachexia,septicshock,andautoimmunedisorders.TNF-aisproducedbyactivatedmacrophagesandothercelltypesincludingTandBcells,NKcells,LAKcells,astrocytes,endothelialcells,smoothmusclecellsandsometumorcells(1-4.
RatTNF-acDNAencodesa235aminoacid(aaresiduetypeIImembraneprotein(5.The156aaresiduesolubleTNF-aisreleasedfromtheC-terminusofthe
membrane-anchoredTNF-abyTNF-a-convertingenzyme(TACE,amatrixmetalloprotease(6,7.Themembrane-anchoredformofTNF-ahasbeenshowntohavelyticactivityandmayalsoplayanimportantroleinintercellularcommunication(8.ThebiologicallyactiveTNF-ahasbeenshowntoexistasatrimer(9,10.
TwodistinctTNFreceptors,referredtoastypeI(ortypeBorp55andtypeII(ortypeAorp75,thatspecificallybindTNF-aandTNF-bwithequalaffinityhavebeenidentified(11,12.ThetwoTNFreceptorstransducesignalsindependentlyofoneanother.TheaminoacidsequenceoftheextracellulardomainsofthetworeceptorsarehomologousandbothreceptorsaremembersoftheTNFreceptorfamilywhichalsoincludetheNGFreceptor,fasantigen,CD27,CD30,andCD40.Theintracellulardomainsofthetworeceptorsareapparentlyunrelated,suggestingthatthetworeceptorsemploydifferentsignaltransductionpathways.Solubleformsofbothtypesofreceptorshavebeenfoundinhumanserumandurine(13-15.ThesesolublereceptorsarecapableofneutralizingthebiologicalactivitiesoftheTNFsandmayservetomodulatetheactivitiesofTNF.
TheQuantikineRatTNF-aImmunoassayisa4.5hoursolidphaseELISAdesignedtomeasureratTNF-alevelsincellculturesupernates,serum,andplasma.Itcontains
E.coli-expressedrecombinantratTNF-aandantibodiesraisedagainsttherecombinantfactor.ThisimmunoassayhasbeenshowntoquantitatetherecombinantratTNF-aaccurately.ResultsobtainedusingnaturalratTNF-ashoweddoseresponsecurvesthatwereparalleltothestandardcurvesobtainedusingtherecombinantkitstandards.TheseresultsindicatethattheQuantikineRatTNF-aImmunoassaykitcanbeusedtodeterminerelativemassvaluesfornaturalratTNF-a.
PRINCIPLEOFTHEASSAY
Thisassayemploysthequantitativesandwichenzymeimmunoassaytechnique.AmonoclonalantibodyspecificforratTNF-ahasbeenpre-coatedontoamicroplate.Standards,Control,andsamplesarepipettedintothewellsandanyratTNF-apresentisboundbytheimmobilizedantibody.Afterwashingawayanyunboundsubstances,anenzyme-linkedpolyclonalantibodyspecificforratTNF-aisaddedtothewells.Followingawashtoremoveanyunboundantibody-enzymereagent,asubstratesolutionisaddedtothewells.TheenzymereactionyieldsablueproductthatturnsyellowwhentheStopSolutionisadded.TheintensityofthecolormeasuredisinproportiontotheamountofratTNF-aboundintheinitialstep.Thesamplevaluesarethenreadoffthestandardcurve.
LIMITATIONSOFTHEPROCEDURE
·FORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.
·Thekitshouldnotbeusedbeyondtheexpirationdateonthekitlabel.
·Donotmixorsubstitutereagentswiththosefromotherlotsorsources.
·Ifsamplesgeneratevalueshigherthanthehigheststandard,furtherdilutethesampleswithCalibratorDiluentandrepeattheassay.
·Anyvariationinoperator,pipettingtechnique,washingtechnique,incubationtimeor
temperature,andkitagecancausevariationinbinding.
·Thisassayisdesignedtoeliminateinterferencebysolublereceptors,bindingproteins,andotherfactorspresentinbiologicalsamples.UntilallfactorshavebeentestedintheQuantikineImmunoassay,thepossibilityofinterferencecannotbeexcluded.
PRECAUTION
TheStopSolutionprovidedwiththiskitisanacidsolution.Weareye,hand,face,andclothingprotectionwhenusingthismaterial.
TECHNICALHINTS
·Whenmixingorreconstitutingproteinsolutions,alwaysavoidfoaming.
·Toavoidcross-contamination,changepipettetipsbetweenadditionsofeachstandardlevel,betweensampleadditions,andbetweenreagentadditions.Also,useseparate
reservoirsforeachreagent.
·Whenusinganautomatedplatewasher,addinga30secondsoakperiodfollowingtheadditionofwashbuffer,and/orrotatingtheplate180degreesbetweenwashstepsmayimproveassayprecision.
·Forbestresults,pipettereagentsandsamplesintothecenterofeachwell.
·Itisrecommendedthatthesamplesbepipettedwithin15minutes.
·Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.
·SubstrateSolutionshouldremaincolorlessuntiladdedtotheplate.KeepSubstrate
Solutionprotectedfromlight.SubstrateSolutionshouldchangefromcolorlessto
gradationsofblue.
·StopSolutionshouldbeaddedtotheplateinthesameorderastheSubstrateSolution.
ThecolordevelopedinthewellswillturnfrombluetoyellowuponadditionoftheStopSolution.
MATERIALSPROVIDED
DescriptionPart#Cat.#
RTA00
Cat.#
SRTA00
RatTNF-aMicroplates-96wellpolystyrenemicroplates
(12stripsof8wellscoatedwithamonoclonalantibodyspecific
forratTNF-a.
8906822plates6plates
RatTNF-aConjugate-23mL/vialofapolyclonalantibody
againstratTNF-aconjugatedtohorseradishperoxidasewith
preservatives.
8926681vial3vials
RatTNF-aStandard-1.6ng/vialofrecombinantratTNF-aina
bufferedproteinbasewithpreservatives;lyophilized.8906843vials9vialsRatTNF-aControl-RecombinantratTNF-ainabuffered
proteinbasewithpreservatives;lyophilized.Theconcentration
rangeofratTNF-aafterreconstitutionisshownontheviallabel.
TheassayvalueoftheControlshouldbewithintherange
specifiedonthelabel.
8906853vials9vials
AssayDiluentRD1-41-12.5mL/vialofabufferedproteinbase
withpreservatives.8955141vial3vialsCalibratorDiluentRD5-17-21mL/vialofabufferedprotein
basewithpreservatives.8955122vials6vialsWashBufferConcentrate-50mL/vialofa25-foldconcentrated
solutionofabufferedsurfactantwithpreservative.8950241vial3vialsColorReagentA-12.5mL/vialofstabilizedhydrogenperoxide.8950001vial3vialsColorReagentB-12.5mL/vialofstabilizedchromogen
(tetramethylbenzidine.8950011vial3vialsStopSolution-23mL/vialofadilutedhydrochloricacidsolution.8951741vial3vialsPlateCovers-Adhesivestrips.___8strips24stripsRTA00containssufficientmaterialstorunELISAsontwo96wellplates.
SRTA00(SixPakcontainssufficientmaterialstorunELISAsonsix96wellplates.
ThiskitisalsoavailableinaPharmPak(R&DSystems,Catalog#PRTA00.PharmPakscontainsufficientmaterialstorunELISAson50microplates.Specificvialcountsofeachcomponentmayvary.Pleaserefertotheliteratureaccompanyingyourorderforspecificvialcounts.
*Providedthisiswithintheexpirationdateofthekit.
OTHER
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