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FORRESEARCHUSEONLY.
NOTFORUSEINDIAGNOSTICPROCEDURES.
RatKidneyinjurymolecule1(Kim-1)ELISAKitinstruction
Intendeduse
ThisKim-1ELISAkitisintendedLaboratoryforResearchuseonlyandisnotforuseindiagnosticortherapeuticprocedures.TheStopSolutionchangesthecolorfrombluetoyellowandtheintensityofthecolorismeasuredat450nmusingaspectrophotometer.InordertomeasuretheconcentrationofKim-1inthesample,thisKim-1ELISAKitincludesasetofcalibrationstandards.ThecalibrationstandardsareassayedatthesametimeasthesamplesandallowtheoperatortoproduceastandardcurveofOpticalDensityversusKim-1concentration.TheconcentrationofKim-1inthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.
Samplecollectionandstorages
Serum-Useaserumseparatortubeandallowsamplestoclotfor30minutesbeforecentrifugationfor10minutesatapproximately3000×g.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20℃or-80℃.Avoidrepeatedfreeze-thawcycles
Plasma-CollectplasmausingEDTAorheparinasananticoagulant.Centrifugesamplesfor30minutesat3000×gat2-8℃within30minutesofcollection.Storesamplesat-20℃or-80℃.Avoidrepeatedfreeze-thawcycles.
Cellculturesupernatesandotherbiologicalfluids-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20℃or-80℃.Avoidrepeatedfreeze-thawcycles.
Note:Thesamples首lebecentrifugateddequatelyandnohemolysisorgranulewasallowed.
Materialsrequiredbutnotsupplied
1.Standardmicroplatereader(450nm)
2.PrecisionpipettesandDisposablepipettetips.
3.37℃incubator
Precautions
1.Donotsubstitutereagentsfromonekittoanother.Standard,conjugateandmicroplatesarematchedforoptimalperformance.Useonlythereagentssuppliedbymanufacturer.
2.Donotremovemicroplatefromthestoragebaguntilneeded.Unusedstrips首ldbestoredat2-8°Cintheirpouchwiththedesiccantprovided.
3.Mixallreagentsbeforeusing.
Removeallkitreagentsfromrefrigeratorandallowthemtoreachroomtemperature(20-25°C)
MaterialssuppliedName
96determinations
48determinations
Microelisastripplate
12*8strips
12*4strips
Standard
0.3ml
0.3ml
Samplediluent
6.0ml
3.0ml
HRP-Conjugatereagent
10.0ml
5.0ml
20XWashsolution
25ml
15ml
ChromogenSolutionA
6.0ml
3.0ml
ChromogenSolutionB
6.0ml
3.0ml
StopSolution
6.0ml
3.0ml
Closureplatemembrane
2
2
Usermanual
1
1
Sealedbags
1
1
Note:Standardconcentrationwasfollowedby:
160、80、40、20、10、5pg/mL.
Reagentpreparation
20×washsolution:DilutewithDistilledordeionizedwater1:20.
Assayprocedure
1.Prepareallreagentsbeforestartingassayprocedure.ItisrecommendedthatallStandardsandSamplesbeaddedinduplicatetotheMicroelisaStripplate.
2.Addstandard:SetStandardwells,testingsamplewells.Addstandard50μltostandardwell.
3.AddSample:Addtestingsample10μlThenaddsamplediluent40μltotestingsamplewell;Blankwelldoesn’taddanyting.
4.Add100μlofHRP-conjugatereagenttoeachwell,coverwithanadhesivestripandincubatefor60minutesat37°C.
5.Aspirateeachwellandwash,repeatingtheprocessfourtimesforatotaloffivewashes.Washbyfil领eachwellwithWashSolution(400μl)usingasquirtbottle,manifolddispenserorautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashSolutionbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.
6.AddchromogensolutionA50μlandchromogensolutionB50μltoeachwell.Gentlymixandincubatefor15minutesat37°C.Protectfromlight.
7.Add50μlStopSolutiontoeachwell.Thecolorinthewells首ldchangefrombluetoyellow.Ifthecolorinthewellsisgreenorthecolorchangedoesnot
appearuniform,gentlytaptheplatetoensurethoroughmixing.8.ReadtheOpticalDensity(O.D.)at450nmusingamicrotiterplatereaderwithin15minutes.
Calculationofresults
1.Thisstandardcurveisusedtodeterminetheamountinanunknownsample.ThestandardcurveisgeneratedbyplottingtheaverageO.D.(450nm)obtainedforeachofthesixstandardconcentrationsonthevertical(Y)axisversusthecorrespondingconcentrationonthehorizontal(X)axis.
2.First,calculatethemeanO.D.valueforeachstandardandsample.AllO.D.values,aresubtractedbythemeanvalueofthezerostandardbeforeresultinterpretation.Constructthestandardcurveusinggraphpaperorstatisticalsoftware.
3.Todeterminetheamountineachsample,firstlocatetheO.D.valueontheY-axisandextendahorizontallinetothestandardcurve.Atthepointofintersection,drawaverticallinetotheX-axisandreadthecorrespondingconcentration.
4.Anyvariationinoperator,pipettingandwashingtechnique,incubationtimeortemperature,andkitagecancausevariationinresult.Eachuser首ldobtaintheirownstandardcurve.
5.Thesensitivitybythisassayis1.0pg/mL.
6.Standardcurve
Storage：2-8℃.
validity：sixmonths.
FORRESEARCHUSEONLY;NOTFORTHERAPEUTICORDIAGNOSTICAPPLICATIONS!PLEASEREADTHROUGHENTIREPROCEDUREBEFOREBEGINNING!
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