CatNo.eia05889Go
GoatInterleukin10(IL-10)ELISAKit
Dearcustomers,thankyouforchoosingourproducts.ThisproductissuitableforinvitroqualitativedetectionofGoatserum,plasmaorcellculturesupernatantandorganizationsinthenaturalandrecombinantIL-10concentration.Detectionofotherspecialsamplepleasecontactourtechnicalsupport.Thekitisforresearchuseonly.Pleasereadtheinstructionscarefullybeforeusingandcheckthekitcomponents.Ifyouhaveanyquestions,pleasecontactSANCHEZINCYouwillgetourfullrangeofservices.
ThiskitemploysDoubleAntibodySandwichTechnique.TheprincipleofDoubleAntibodySandwichisbasedoncharacteristicsofthetestedantigenwithmorethantwovalanceswhichcanidentifycoatedantibodyanddetectionantibodyatsametime.Thespecificprocessisasfollows:
1.Connectthespecificantibodiesandsolidphasecarrierstoformimmobilizedantibodies.Washoutuncombinedantibodiesandimpurities.Sealtherestbindingsiteswithirrelevantproteins.
2.Joinundertestwithimmobilizedantibodiesforcontactreaction.Afterawhile,combineantigensinandantibodiesoncarriersintotheantigenscomplex.Washoutuncombinedantibodiesandimpurities.
3.Addbiotinlabelingantibodiestocombinewiththeantigensonimmunecomplexes.Washouttheuncombinedbiotinlabelingantibodiesthoroughly.Theenzymeamountonthecarrierisnowpositivelyrelatedtotheamountofthetestedsubstanceinspecimens.
4.Addhorseradishperoxidasetolabeltheavidinsandincorporatethemwiththebiotinlabelingantibodies.Washouttheincorporatedenzyme
Markersthoroughly.Theenzymeamountonthecarrierisnowpositivelyrelatedtotheamountofthetestedsubstanceinspecimens.
5.Addsubstratesforcoloring,andcomputetheconcentrationofspecimens.
Note:anantibodymoleculecanbemarkedonseveralbiotinmoleculesandabiotinmoleculecanbeconnectedwithaHRP-AvidintoformnumbersofhorseradishperoxidasescombiningwithantibodieswhichshowshighersensitivityandamplificationeffectcomparingwithtraditionaldirectHRP-Antibodies.
【DetectionprincipleofGoatInterleukin10(IL-10)ELISAkit】
Thisexperimentusedouble-sandwichelisatechniqueandtheELISAKitprovidedistypical.Thepre-coatedantibodyisGoatIL-10monoclonalantibodyandthedetectingantibodyispolyclonalantibodywithbiotinlabeled.SamplesandbiotinlabelingantibodyareaddedintoELISAplatewellsandwashedoutwithPBSorTBS.ThenAvidin-peroxidaseconjugatesareaddedtoELISAwellsinorder;UseTMBsubstrateforcoloringafterreactantthoroughlywashedoutbyPBSorTBS.TMBturnsintoblueinperoxidasecatalyticandfinallyturnsintoyellowundertheactionofacid.Thecolordepthandthetestingfactorsinsamplesarepositivelycorrelated.
【Kitcomposition】
name 96Tests 48TestsStorage
1．antibodyprecoatedplate 8×12 8×64/-20℃
2．GoatIL-10Standards 2vial 1vial4/-20℃
3．Biotinylatedantibody(1:100) 1vial 1vial4/-20℃
4．Enzymeconjugate(1:100)1vial 1vial4/-20℃
5．Enzymediluent 1vial 1vial 4/-20℃
6．antibodydiluent 1vial 1vial4/-20℃
7．Standarddiluent 1vial1vial 4/-20℃
8．Samplediluent 1vial 1vial 4/-20℃
9．Washingbuffer(1:25) 1vial1vial 4/-20℃
10．ColourReagentA 1vial1vial 4/-20℃
11．ColourReagentB1vial1vial 4/-20℃
12．ColourReagentC 1vial1vial 4/-20℃
13．Instruction 1set1set RT
Note：
RT：Roomtemperature
Standard：Frozendried
ColourReagentA:Avoidlight
【Necessaryfortestingtheirowntestfacilitiesandequipment】
1.Microplatereader(450nmdetectionwavelengthfilter,570nmor630nmcorrectionwavelengthfilters).
2.Washer(adjustableamountofliquidinjectiontoensurethateachwell350μllotionwithoutoverflow).
3.Cleanbenches,biologicalsafetycabinets,fumehoods.
4.High-precisionsingle-channeldispenser(range0.5-10μl-20μl,20-200μl,200-1000μl).
5.High-precisionmulti-channelplusliquid(8or12,therangeof50-300μlof).
6.37℃incubator.
7.Lowtemperaturecentrifuge.
8.Refrigerators(4℃,-20℃,-86℃).
9.Analyticalbalance.
10.Scissors,tweezers,pliers,andsoon.
11.Swirlmixingdevice,low-frequencyoscillator,andsoon.
【Necessaryfortestingtheirowntestingsuppliesandreagents】
1.Centrifugetube(capacityof1.5ml,5ml,andsoon).
2.Disposabletip(rangeof0.5-10μl-20μl,20-200μl,200-1000μl).
3.Purewaterordistilledwater.
4.Coordinatepaper.
5.Absorbentpaper.
6.EDTA,sodiumcitrate,heparin
【SamplecollectionNote】
1.Thetubeforbloodcollectionshouldbefreeofpyrogenandendotoxin
2.Hemolysisandhyperlipidemiaspecimenscannotbeusedtoextractedserumandplasma.
3. Thesamplesshouldappearclearandtransparent.Andallthesuspensionshouldberemovedthroughcentrifugation.
4. Ifcollectedsamplesarenottimelydetected,theyshouldbedividedaccordingtosingleusageamountandfrozenreservedinrefrigeratorat-20-80℃,avoidingtherepeatedfreeze-thaw.
5. Accordingtotheactualsituationofthesamples,makepropermultipledilutions(Pre-experimentisstronglyrecommendedinordertoconfirmthedilutionratio)
6. Collectspecimensandtrytogaindoubledosagetoavoidspecimensshortageforrepeatedassaysincasethatfailureinone-assaydelaysexperimentalprocess.
7. Doprotectivemeasureswhencollectingspecimens(e.g.wearinggloves,respirator,respirator,etc.),awareofthepotentialriskinallspecimens.
8. Specimenprocessingshouldbeinsidethebiologicalsafetycabinet.Ensureproperuseofthebiologicalsafetycabinet.
【Measuresforthesamples】
1. Serum:Putthecollectedwholebloodinrefrigeratorat4℃forthenight.Thencentrifugeitfor10minat1000-3000rpm.Takesupernatanttestedimmediatelyorputsamplesat-20℃(for1-3months)or-80℃(for1-3months)forstorage.
2. Plasma:TakeEDTA,sodiumcitrateandheparinasanticoagulant.Addtheplasmaandmixthemwell.Centrifugemixturefor10minat1000-3000rpm.Takesupernatanttestedimmediatelyorputsamplesat-20℃(for1-3months)or-80℃(for1-3months)forstorage.
3.Tissuehomogenate:Taketissueslicesandwashthemoutin0.01MPBS;Addtissueproteinextractionreagentaccordingtoproportionof1G:5-10mlandmixtheminicewater.Afterbeingblended,mixtureshallbecentrifugedfor10minat5000-10000rpm.Takesupernatanttestedimmediatelyorputthemat-20℃(for1-3months)or-80℃(for1-3months)forstorage.
4. Cellculture:Takecentrifugationfor10minat1000-3000rpm.Takesupernatanttestedimmediatelyorputsamplesat-20℃(for1-3months)or-80℃(for1-3months)forstorage.
5.Forurine,ascites,cerebrospinalfluid,etc:akecentrifugationfor10minat1000-3000rpm.Takesupernatanttestedimmediatelyorputsamplesat-20℃(for1-3months)or-80℃(for1-3months)forstorage.
Note:Thegeneralprinciplesofthesampledilution
Theusershouldrefertothereferencestoknowtheprobablecontentofthesamplesbeforedecidetodilutethesamples,andthedilutedcontentofthesamplemustbeinthebestdetectionrangeofthegivenELISAKits.Thedilutionofthesampleshouldberecordedindetail.
【Note】
1. Thekitshouldbekeptat2-8℃beforebeingused.Excepttheredissolvedstandardsamples,otherIngredientsmustnotbefrozen.
2.FortheconcentratedbiotinylatedGoatIL-10antibody,theconcentratedenzyme-conjugateshavesmallsize.Bumpingorpotentialinversionofthetubesduringtransportationmaycausetheliquidstickingtowallorcap.Thus,thetubesshouldbeshakenmanuallyorcentrifugedfor1minat1000rpmtoshakeofftheadherentliquiddowntothetubebottom.
3. Concentratedwashingbuffermaycrystallizealittle.Usewaterbathtohelpthedissolutionduringdilutingprocess.Thecrystalsmustbetotallydissolvedwhenpreparingwashingbuffer.
4.Duringtestingprocess,theGoatIL-10lyophilizedstandardsampleshallbesingle-useandmustnotbedivided.Thesamplewillquicklyinactivateafterbeingdissolvedbecauseofitslowerconcentration.
5.Operationshouldbestrictlyinaccordancewiththeinstructions.Mixedusageofcomponentswithdifferentbatchnumberinthisreagentisnotallowed
6. Ensurethereagentwellmixedbythespiralhybridinstrument.Forthereagentinthemicroplate,adequatemixingisparticularlyimportanttotestresult.Soit’sbettertoemploythemicro-oscillator(atthelowestfrequency).Ifthereisnotmicro-oscillator,shakethemicroplatemanuallyfor1min,slightlyaslikeacircularmovementtomakesurereactionliquidinmicroplatewellmixed
7. ELISAforexperimentshouldbestrictlyoperatedaccordingtomanualstandardandfullypreheatedbeforehand.
8. Duringenzymeimmunoassay,thereshouldbemultipleporeswhentestingGoatIL-10standardsamples
9. Puttheunusedmicroplatesintorawfoilbagat2-8℃forstorage.
10.Chromogenreagentissensitivetolight.Thereforeitshouldbefreeofbeingexposedtolight.
11.Kitsoutofvalidityshouldnotbeusedinexperiment.
12.ThedeterminationoftestresultsmustbesubjecttoELISA’sreadings.Whenusingdual-wavelengthfortest,thewavelengthshouldbesetat450nmand630nmrespectively.
13.Allthesamples,washingliquidandwastesshouldbetreatedasbiowaste.ColourReagentCshouldbe2Msulfuricacidandpayattentiontosafetywhenitisused.
14.Sample-addingateverystepshouldbetakenbyaddinginstrument.Calibrateaccuracyoftheaddingdevicetoavoidexperimenterror.Thetimeofsinglesample-addingshouldbecontrolledwithin5min.Justincaseofexceedingsamples,thevolleyforsample-addingisproposed.
15.Adhesiveclosuresdonotreuseoraccordingtotheexperimentsneedtobecropped.Stickastripofadhesivetocompaction
16.Testdeterminationandstandardcurveshouldbemadeatsametimeineveryexperiment,sotherebetterbemultiplepores.Ifthecontentoftestsampleweretoohigh(ODvalueofthesampleishigherthanthatofsamplewellmaximumconcentration),dilutetocertainmultiplebysamplediluents(ntimes),thentesttheresultandmultiplyitbydilutionratioswhenmakingcalculation.
17.ThesamplecontainingNaN3can’tbetestedbecauseNaN3inhibittheactivitiesofhorseradishperoxidase(HRP).
18.Whenwashingboardbyplatewasher,thevolumeofliquidinjectiontoeachwellshouldbemorethan350μl.Checkifthesamplingheadisjammed.YetthewaterabsorptionmaterialwithPaperScrapshouldbecautiouslyusedwhilewashingboardmanually,freeofthereactionbetweenexogenousperoxidaseanaloguesandchromogenreagents.
19.AfterthereactionbeingterminatedbyColourReagentC,readODwithin10min.
20.Duringmultipleporesexperiment,thecalculationresultshallbemeanvalue.
21.Samplehemolysismaycausefalsepositiveresult,sothistestisnotappropriateforsamplehemolysis.
22.Duringtest,thestripsshouldbeputintoclosedboxafteraddingsamplesandthehumidityaroundshouldbekeptatabout60%
23.Itisadvisabletocheckthethermostatbyfrequentcalibrationtokeepitsinsidetemperatureat37℃.Ensuretheexperimentaltemperaturebeingsteady.
24.For48TElisaKit,allcomponentsare50%amountof96T.
【Testpreparation】
1.PleasegettheElisaKitoutofrefrigerator20minutesinadvanceandtaketestwhenitbalancestoroomtemperature.
2.Dilutetheconcentratedwashingsolutionwithdoubledistilledwater(1:25).Puttheunusedback.
3.GoatIL-10standardsample: Adddiluent1.0mlintoGoatIL-10lyophilizedstandardsampleandkeepitstillfor30min.Afterthesamplecompletelydissolved,mixitslightlyandmarklabelonthetube①，thentakedilutionasneeded.(Itisrecommendedtousingfollowingconcentrationvaluetostandardcurve:1000,500,250,125,62.5,31.2,15.6pg/ml).Note:Makesurethelyophilizedstandardcompletelydissolvedandwellmixed.
4.Legendofstandardsampledilutionmethod:Take7cleantubesandlabelthemwith②,③,④,⑤,⑥,⑦,⑧respectively.Add300μlstandardsamplediluentintoeachtube.Pipetteout300μldiluentfromtube①totube②andmixwell.FurtherPipetteout300μldiluentfromtube②totube③,andmixwell.Repeatstepsaboveuptotube⑦.Standardsampledilutionintube⑧isnegativecontrol.
Note:Theredissolvedstandardliquid（1000pg/ml）shallbediscardedandnotnon-reusable.
5.BiotinylatedGoatIL-10antibodyliquid:Referringtoneededamount,employantibodydiluenttodilutetheconcentratedbiotinylatedantibody(1:100)toformbiotinylatedantibodyliquid.Thepreparationshouldbedone30mininadvance.Andit’sonlyforuseonthatday
6.Enzyme-conjugateliquid:Referringtoneededamount,dilutetheconcentratedenzyme-conjugatebyenzyme-conjugatediluent(1:100)toformenzyme-conjugateliquid.Thepreparationshouldbedone30mininadvance.Andit’sonlyforuseonthatday.
7.ColourReagentliquid:PrepareColourReagentliquid30mininadvancewithColourReagentAandColourReagentBbytheproportionof9:1.
【Washingmethod】
1.Automaticplate-washingmachine:Therequiredamountoflotionis350μlandtheinjectionandextractionintervalshouldbe20－30secs.Bewellawareoftheoperationinstructionbeforeputtingthemachineintopractice.
2.Manualplate-washingmachine:add350μllotiontoeachwellandkeepitstillfor30secs.Shakeindividualwellsasdryasyoucanandcleanthemwithabsorbentpaper.Duringtheplate-washingprocess,payattentiontothelotion-addingsteptoavoidcontaminationandwell-jumping..
【Steps】
Read-ifusingtwowavelengthplate,emptyholescannot;ifusingareadsinglewavelengthplate,settheemptyhole,emptyholesinadditiontocolorworkingliquidandTMBterminatesotherthanliquid,withoutanyreagent.
1.Takeoutneededstripsfromziplockbagwhichbalancestoroomtemperature.Theunusedstripsanddesiccantshouldbeputbackintothesealedaluminumfoilbagat2-8℃forstorage.
2.Setasideblankwells(ifdual-wavelengthreadingplateisused,theblankwellscouldbeignored)
3.AddsamplesordifferentconcentrationofGoatIL-10standardsamplestocorrespondingwells(100μlforeachwell),0pg/mlwellshouldbefilledwithstandarddiluent.Sealthereactionwellswithadhesivetapes,hatchinginincubatorat37℃for90min.
4.PreparebiotinylatedGoatIL-10antibodyliquid30mininadvance.
5.WashtheElisaplate3times
6.AddthebiotinylatedGoatIL-10antibodyliquidtoeachwell(100μlforeach).Sealreactionwellswithadhesivetapes, hatching inincubatorat37℃for60min.
7.Prepareenzyme-conjugateliquid30mininadvance.
8.WashtheElisaplate3times
9.Addenzyme-conjugateliquidtoeachwellexceptblankwells(100μlforeach).Sealthereactionwellswith adhesivetapes, hatching inincubatorat37℃for30min.
10.WashtheElisaplate5times.
11.Add100μlColourReagentliquidtoindividualwell(alsointoblankwell),hatchingindarkincubatorat37℃.Whencolorforhighconcentrationofstandardcurvebecomedarkerandcolorgradientappears,thehatchingcanbestopped.Thechromogenicreactionshouldbecontrolledwithin30min.
12.Add100μlColourReagentCtoindividualwell(alsointoblankwell).Mixwell.ReadOD（450nm）within10min.
【Resultdetermination】
1.ODvalueofeachsampleandspecimenshouldminusthatofblankwell(ifnot,thestandardcurveofzerowellshouldintersectatYaxis)
2.Drawstandardcurvemanually.TakeconcentrationvalueofsamplesasabscissaandODreadingsasverticalcoordinate.Usesmoothlinetoconnecteachcoordinatepointofstandardsample.TheconcentrationofsamplescanbefoundbycheckingsampleODreading.Itisrecommendedtoemploytheprofessionalcurvesoftware(e.g.curveexpert1.3)toanalyzeandcomputetheresult.
3.IfthesampleODishigherthantheupperlimitofstandardcurve,thesampleshouldbere-dilutedandtheexperimentrerun.Multiplytheresultbydilutionfactorwhencalculatingtheunknown.
Note:Thischartisonlyforreference.Thecalculationofspecimens’contentshallbesubjecttothestandardcurvemadeforsamplesinsameexperiment.
【Referencecurve】
Note:Thischartisforreferenceonly,shouldbebasedontheteststandarddwarfedstandardcurvetocalculatethespecimencontent.
【Summaryofoperatingprocedures】
Preparereagents,samplesandstandards
Addtothepreparedsampleandthestandardreactionat37℃for90minutes
Washetheplate,addingabiotinylatedantibodyworkingsolution,37℃for60minutes
WashedthreetimestojointheEnzymeworkingsolution,37℃for30minutes
Washerfivetimes,addingtheColourReagentsolution,37℃for
AddtotheColourReagentC
MicroplatereadermeasuredODvalueswithin10minutes
Calculatethefactorcontentofspecimenstested
Kitparameters
【Detectionrange】1000pg/ml-15.6pg/ml
【Sensitivity】theminimumdetectableGoatIL-10upto5pg/ml.
【Specific】Nocross-reactionwithotherfactors.
【IntraassayPrecision】≤8%
【InterassayPrecision】≤12%
【Recovery】70-110percent.
【Storage】-20℃[Short-termshouldbeplaced4℃(suchastwoweeks)]
【Uses】usedinvitroquantitativeanalysisofliquidsamples(Universal).
【Specifications】96T.
【ProductionDate】Seemicrotiterplatesaluminumfoilbagsealingstamp.
【Validity】12months(-20℃).
【Reference】
1.EskdaleJ,KubeD,TeschH,GallagherG(1997).
2.ZdanovA,Schalk-HihiC,GustchinaA,TsangM,WeatherbeeJ,WlodawerA(June1995).
3.PestkaS,KrauseCD,SarkarD,WalterMR,ShiY,FisherPB(2004).
4.SaidE.A.,TrautmannL.,DupuyF.,ZhangY.,AncutaP.,El-FariM.,DouekD.,HaddadE.,andSekalyR.-P.(2010.)
5.LiX,MaiJ,VirtueA,etal.(March2012).
6.SaraivaM,O"GarraA(March2010).
