检测TNF的elisa试剂盒说明书

QuantikineÒ

RatTNF-aImmunoassay

CatalogNumberRTA00

SRTA00

PRTA00

Forthequantitativedeterminationofrattumornecrosisfactoralpha(TNF-a

concentrationsincellculturesupernates,ratserum,andplasma.

Thispackageinsertmustbereadinitsentiretybeforeusingthisproduct.

FORRESEARCHUSEONLY.

NOTFORUSEINDIAGNOSTICPROCEDURES.

TABLEOFCONTENTS

ContentsPage

INTRODUCTION2PRINCIPLEOFTHE

ASSAY..................................3LIMITATIONSOFTHE

PROCEDURE3PRECAUTION..........................................3

TECHNICALHINTS3MATERIALS

PROVIDED....................................4STORAGE5OTHER

SUPPLIESREQUIRED.................................5SAMPLE

COLLECTIONANDSTORAGE6SAMPLE

PREPARATION....................................6REAGENT

PREPARATION7ASSAY

PROCEDURE......................................8PROCEDURE

SUMMARYANDCHECKLIST9CALCULATIONOF

RESULTS.................................10TYPICALDATA10

PRECISION..........................................11RECOVERY11

LINEARITY...........................................12SENSITIVITY12

CALIBRATION.........................................13SAMPLE

VALUES13SPECIFICITY.........................................13

REFERENCES14PLATELAYOUT........................................15

MANUFACTUREDANDDISTRIBUTEDBY:

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Minneapolis,MN55413FAX:(612656-4400

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INTRODUCTION

Tumornecrosisfactoralpha(TNF-a,alsoknownascachectin;andtumornecrosis

factorbeta(TNF-b,alsoknownaslymphotoxin,aretwocloselyrelatedproteins

(approximately34%aminoacidsequenceidentitythatbindtothesamecellsurface

receptorsandshowmanycommonbiologicalfunctions.TNF-aand-bplaycriticalroles

innormalhostresistancetoinfectionandtothegrowthofmalignanttumors,servingas

immunostimulantsandasmediatorsoftheinflammatoryresponse.Over-productionof

TNFs,however,hasbeenimplicatedasplayingaroleinanumberofpathological

conditions,includingcachexia,septicshock,andautoimmunedisorders.TNF-ais

producedbyactivatedmacrophagesandothercelltypesincludingTandBcells,NK

cells,LAKcells,astrocytes,endothelialcells,smoothmusclecellsandsometumorcells

(1-4.

RatTNF-acDNAencodesa235aminoacid(aaresiduetypeIImembraneprotein(5.

The156aaresiduesolubleTNF-aisreleasedfromtheC-terminusofthe

membrane-anchoredTNF-abyTNF-a-convertingenzyme(TACE,amatrix

metalloprotease(6,7.Themembrane-anchoredformofTNF-ahasbeenshowntohave

lyticactivityandmayalsoplayanimportantroleinintercellularcommunication(8.The

biologicallyactiveTNF-ahasbeenshowntoexistasatrimer(9,10.

TwodistinctTNFreceptors,referredtoastypeI(ortypeBorp55andtypeII(or

typeAorp75,thatspecificallybindTNF-aandTNF-bwithequalaffinityhavebeen

identified(11,12.ThetwoTNFreceptorstransducesignalsindependentlyofoneanother.

Theaminoacidsequenceoftheextracellulardomainsofthetworeceptorsare

homologousandbothreceptorsaremembersoftheTNFreceptorfamilywhichalso

includetheNGFreceptor,fasantigen,CD27,CD30,andCD40.Theintracellular

domainsofthetworeceptorsareapparentlyunrelated,suggestingthatthetworeceptors

employdifferentsignaltransductionpathways.Solubleformsofbothtypesofreceptors

havebeenfoundinhumanserumandurine(13-15.Thesesolublereceptorsarecapable

ofneutralizingthebiologicalactivitiesoftheTNFsandmayservetomodulatethe

activitiesofTNF.

TheQuantikineRatTNF-aImmunoassayisa4.5hoursolidphaseELISAdesigned

tomeasureratTNF-alevelsincellculturesupernates,serum,andplasma.Itcontains

E.coli-expressedrecombinantratTNF-aandantibodiesraisedagainstthe

recombinantfactor.Thisimmunoassayhasbeenshowntoquantitatetherecombinantrat

TNF-aaccurately.ResultsobtainedusingnaturalratTNF-ashoweddoseresponsecurves

thatwereparalleltothestandardcurvesobtainedusingtherecombinantkitstandards.

TheseresultsindicatethattheQuantikineRatTNF-aImmunoassaykitcanbeusedto

determinerelativemassvaluesfornaturalratTNF-a.

PRINCIPLEOFTHEASSAY

Thisassayemploysthequantitativesandwichenzymeimmunoassaytechnique.A

monoclonalantibodyspecificforratTNF-ahasbeenpre-coatedontoamicroplate.

Standards,Control,andsamplesarepipettedintothewellsandanyratTNF-apresentis

boundbytheimmobilizedantibody.Afterwashingawayanyunboundsubstances,an

enzyme-linkedpolyclonalantibodyspecificforratTNF-aisaddedtothewells.

Followingawashtoremoveanyunboundantibody-enzymereagent,asubstratesolution

isaddedtothewells.Theenzymereactionyieldsablueproductthatturnsyellowwhen

theStopSolutionisadded.Theintensityofthecolormeasuredisinproportiontothe

amountofratTNF-aboundintheinitialstep.Thesamplevaluesarethenreadoffthe

standardcurve.

LIMITATIONSOFTHEPROCEDURE

·FORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTIC

PROCEDURES.

·Thekitshouldnotbeusedbeyondtheexpirationdateonthekitlabel.

·Donotmixorsubstitutereagentswiththosefromotherlotsorsources.

·Ifsamplesgeneratevalueshigherthanthehigheststandard,furtherdilutethe

sampleswithCalibratorDiluentandrepeattheassay.

·Anyvariationinoperator,pipettingtechnique,washingtechnique,incubationtime

or

temperature,andkitagecancausevariationinbinding.

·Thisassayisdesignedtoeliminateinterferencebysolublereceptors,binding

proteins,andotherfactorspresentinbiologicalsamples.Untilallfactorshavebeentested

intheQuantikineImmunoassay,thepossibilityofinterferencecannotbeexcluded.

PRECAUTION

TheStopSolutionprovidedwiththiskitisanacidsolution.Weareye,hand,face,

andclothingprotectionwhenusingthismaterial.

TECHNICALHINTS

·Whenmixingorreconstitutingproteinsolutions,alwaysavoidfoaming.

·Toavoidcross-contamination,changepipettetipsbetweenadditionsofeach

standardlevel,betweensampleadditions,andbetweenreagentadditions.Also,use

separate

reservoirsforeachreagent.

·Whenusinganautomatedplatewasher,addinga30secondsoakperiodfollowing

theadditionofwashbuffer,and/orrotatingtheplate180degreesbetweenwashsteps

mayimproveassayprecision.

·Forbestresults,pipettereagentsandsamplesintothecenterofeachwell.

·Itisrecommendedthatthesamplesbepipettedwithin15minutes.

·Toensureaccurateresults,properadhesionofplatesealersduringincubationsteps

isnecessary.

·SubstrateSolutionshouldremaincolorlessuntiladdedtotheplate.KeepSubstrate

Solutionprotectedfromlight.SubstrateSolutionshouldchangefromcolorlessto

gradationsofblue.

·StopSolutionshouldbeaddedtotheplateinthesameorderastheSubstrate

Solution.

Thecolordevelopedinthewellswillturnfrombluetoyellowuponadditionofthe

StopSolution.

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/vialofa

bufferedprotein

basewithpreservatives.8955122vials6vialsWashBufferConcentrate-50mL/vial

ofa25-foldconcentrated

solutionofabufferedsurfactantwithpreservative.8950241vial3vialsColor

ReagentA-12.5mL/vialofstabilizedhydrogenperoxide.8950001vial3vialsColor

ReagentB-12.5mL/vialofstabilizedchromogen

(tetramethylbenzidine.8950011vial3vialsStopSolution-23mL/vialofadiluted

hydrochloricacidsolution.8951741vial3vialsPlateCovers-Adhesivestrips.___8

strips24stripsRTA00containssufficientmaterialstorunELISAsontwo96wellplates.

SRTA00(SixPakcontainssufficientmaterialstorunELISAsonsix96wellplates.

ThiskitisalsoavailableinaPharmPak(R&DSystems,Catalog#PRTA00.

PharmPakscontainsufficientmaterialstorunELISAson50microplates.Specificvial

countsofeachcomponentmayvary.Pleaserefertotheliteratureaccompanyingyour

orderforspecificvialcounts.

*Providedthisiswithintheexpirationdateofthekit.

OTHERSUPPLIESREQUIRED

·Microplatereadercapableofmeasuringabsorbanceat450nm,withthecorrection

wavelengthsetat540nmor570nm.

·Pipettesandpipettetips.

·Deionizedordistilledwater.

·Squirtbottle,manifolddispenser,orautomatedmicroplatewasher.

·100mLand1000mLgraduatedcylinders.

·Polypropylenetesttubesfordilution.

SAMPLECOLLECTIONANDSTORAGE

CellCultureSupernates-Removeparticulatesbycentrifugationandassay

immediatelyoraliquotandstoresamplesat£-20°C.Avoidrepeatedfreeze-thawcycles.

Serum-Allowbloodsamplestoclotfor2hoursatroomtemperaturebefore

centrifugingfor20minutesat1000xg.Removeserumandassayimmediatelyoraliquot

andstoresamplesat£-20°C.Avoidrepeatedfreeze-thawcycles.

Plasma-CollectplasmausingEDTAorheparinasananticoagulant.Centrifugefor

20minutesat1000xgwithin30minutesofcollection.Assayimmediatelyor

aliquotandstoresamplesat£-20°C.Avoidrepeatedfreeze-thawcycles.

Note:Grosslyhemolyzedorlipemicsamplesmaynotbesuitableformeasurementof

ratTNF-awiththisassay.

SAMPLEPREPARATION

Ratserumandplasmasamplesrequirea2-folddilutionintoCalibratorDiluentRD5-

17priortoassay.Asuggested2-folddilutionis75mLsample+75mLCalibrator

DiluentRD5-17.Mixwell.

Ratcellculturesupernatesamplesrequirea3-folddilutionintoCalibratorDiluent

RD5-17priortoassay.Asuggested3-folddilutionis50mLsample+100mLCalibrator

DiluentRD5-17.Mixwell.

REAGENTPREPARATION

Bringallreagentstoroomtemperaturebeforeuse.

RatTNF-aKitControl-ReconstitutetheKitControlwith1.0mLdeionizedor

distilledwater.AssaytheControlundiluted.

WashBuffer-Ifcrystalshaveformedintheconcentrate,warmtoroomtemperature

andmixgentlyuntilthecrystalshavecompletelydissolved.ToprepareenoughWash

Bufferforoneplate,add25mLWashBufferConcentrateintodeionizedordistilled

watertoprepare625mLofWashBuffer.

SubstrateSolution-ColorReagentsAandBshouldbemixedtogetherinequal

volumeswithin15minutesofuse.Protectfromlight.100mLoftheresultantmixtureis

requiredperwell.RatTNF-aStandard-ReconstitutetheratTNF-aStandardwith2.0mL

ofCalibrator

DiluentRD5-17.Donotsubstituteotherdiluents.Thisreconstitutionproducesa

stocksolutionof800pg/mL.Allowthestandardtositforaminimumof5minuteswith

gentlemixingpriortomakingdilutions.

Usepolypropylenetubes.Pipette200mLofCalibratorDiluentRD5-17intoeach

tube.Usethestocksolutiontoproduceadilutionseries(below.Mixeachtube

thoroughlybeforethenexttransfer.TheundilutedratTNF-aStandardservesasthehigh

standard(800pg/mL.CalibratorDiluentRD5-17servesasthezerostandard(0pg/mL.

ASSAYPROCEDURE

Bringallreagentsandsamplestoroomtemperaturebeforeuse.Itis

recommendedthatallsamples,standards,andcontrolbeassayedinduplicate.

1.Preparereagents,workingstandards,control,andsamplesasdirectedinthe

previous

sections.

2.Removeexcessmicroplatestripsfromtheplateframe,returnthemtothefoil

pouch

containingthedesiccantpack,andreseal.

3.Add50mLofAssayDiluentRD1-41toeachwell.

4.Add50mLofStandard,Control,orsample*toeachwell.Mixbygentlytapping

theplate

framefor1minute.Coverwiththeadhesivestripprovided.Incubatefor2hoursat

roomtemperature.Aplatelayoutisprovidedtorecordstandardsandsamplesassayed.

5.Aspirateeachwellandwash,repeatingtheprocessfourtimesforatotaloffive

washes.

WashbyfillingeachwellwithWashBuffer(400mLusingasquirtbottle,manifold

dispenser,orautowasher.Completeremovalofliquidateachstepisessentialto

goodperformance.Afterthelastwash,removeanyremainingWashBufferbyaspirating

orbyinvertingtheplateandblottingitagainstcleanpapertowels.

6.Add100mLofRatTNF-aConjugatetoeachwell.Coverwithanewadhesive

strip.

Incubatefor2hoursatroomtemperature.

7.Repeattheaspiration/washasinstep5.

8.Add100mLofSubstrateSolutiontoeachwell.Incubatefor30minutesatroom

temperature.Protectfromlight.

9.Add100mLofStopSolutiontoeachwell.Gentlytaptheplatetoensurethorough

mixing.

10.Determinetheopticaldensityofeachwellwithin30minutes,usingamicroplate

reader

setto450nm.Ifwavelengthcorrectionisavailable,setto540nmor570nm.If

wavelengthcorrectionisnotavailable,subtractreadingsat540nmor570nmfrom

thereadingsat450nm.Thissubtractionwillcorrectforopticalimperfectionsintheplate.

Readingsmadedirectlyat450nmwithoutcorrectionmaybehigherandless

accurate.*SamplesrequiredilutionasdirectedintheSamplePreparationsection.

PROCEDURESUMMARYANDCHECKLIST

CALCULATIONOFRESULTS

Averagetheduplicatereadingsforeachstandard,control,andsampleandsubtract

theaveragezerostandardopticaldensity.

Createastandardcurvebyreducingthedatausingcomputersoftwarecapableof

generatingafourparameterlogistic(4-PLcurve-fit.Asanalternative,constructa

standardcurvebyplottingthemeanabsorbanceforeachstandardonthey-axisagainst

the

concentrationonthex-axisanddrawabestfitcurvethroughthepointsonthegraph.

ThedatamaybelinearizedbyplottingthelogoftheratTNF-aconcentrationsversusthe

logoftheO.D.andthebestfitlinecanbedeterminedbyregressionanalysis.This

procedurewillproduceanadequatebutlessprecisefitofthedata.

Becausesampleshavebeendiluted,theconcentrationreadfromthestandardcurve

mustbemultipliedbythedilutionfactor.

TYPICALDATA

Thisstandardcurveisprovidedfordemonstrationonly.Astandardcurveshouldbe

generatedforeachsetofsamplesassayed.

(pg/mL

012.52550100200400800O.D.0.0340.0340.0850.0800.1280.1270.2140.2160.3830.3

720.6920.6981.2181.2222.0231.988

Average0.0340.0820.1280.2150.3780.6951.2202.006

Corrected

___0.0480.0940.1810.3440.6611.1861.972

PRECISION

Intra-assayPrecision(Precisionwithinanassay

Threesamplesofknownconcentrationweretestedtwentytimesononeplateto

assessintra-assayprecision.

Inter-assayPrecision(Precisionbetweenassays

Threesamplesofknownconcentrationweretestedintwentyassaystoassessinter-

assayprecision.

Intra-assayPrecisionInter-assayprecision

Sample123123

n202020202020

Mean(pg/mL6523259363246656

Standard

6.123.657.6

deviation

CV(%

RECOVERY

TherecoveryofratTNF-aspikedtothreelevelsthroughouttherangeoftheassayin

variousmatriceswasevaluated.

*SamplesweredilutedasdirectedintheSamplePreparationsection.

LINEARITY

Toassessthelinearityoftheassay,samplesspikedwithvariousconcentrationsof

ratTNF-aweredilutedwithCalibratorDiluentRD5-17andthenassayed.Resultsfrom

typicalsampledilutionsareshown.

*Samplesweredilutedpriortoassay,asdirectedintheSamplePreparation

section.

SENSITIVITY

Theminimumdetectabledose(MDDofratTNF-aistypicallylessthan5pg/mL.

TheMDDwasdeterminedbyaddingtwostandarddeviationstothemeanoptical

densityvalueof20zerostandardreplicatesandcalculatingthecorresponding

concentration.

CALIBRATION

ThisimmunoassayiscalibratedagainstahighlypurifiedE.coli-expressed

recombinantratTNF-aproducedatR&DSystems.TherecombinantN-methionylform

ofratTNF-acontains157aminoacidresiduesandhasapredictedmolecularmassof17

kDa.

Basedontotalaminoacidanalysis,theabsorbanceofa1mg/mLsolutionoftheE.

coli-expressedrecombinantratTNF-aat280nmwasdeterminedtobe1.33A.U.

SAMPLEVALUES

Serum/Plasma-Fortyindividualratserumsamplesandthirteenindividualrat

plasmasampleswereevaluatedfordetectablelevelsofratTNF-ainthisassay.All

samplesmeasuredles