大鼠尿素氮（BUN）ELISA试剂盒英文版

FORRESEARCHUSEONLY.

NOTFORUSEINDIAGNOSTICPROCEDURES.

RatUreanitrogen(BUN)ELISAKitinstruction

Intendeduse

ThisBUNELISAkitisintendedLaboratoryforResearchuseonlyandisnotforuseindiagnosticortherapeuticprocedures.TheStopSolutionchangesthecolorfrombluetoyellowandtheintensityofthecolorismeasuredat450nmusingaspectrophotometer.InordertomeasuretheconcentrationofBUNinthesample,thisBUNELISAKitincludesasetofcalibrationstandards.ThecalibrationstandardsareassayedatthesametimeasthesamplesandallowtheoperatortoproduceastandardcurveofOpticalDensityversusBUNconcentration.TheconcentrationofBUNinthesamplesisthendeterminedbycomparingtheO.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:

24、12、6、3、1.5、0.75mmol/L.

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.Thesensitivitybythisassayis0.1mmol/L.

6.Standardcurve

Storage：2-8℃.

validity：sixmonths.

FORRESEARCHUSEONLY;NOTFORTHERAPEUTICORDIAGNOSTICAPPLICATIONS!PLEASEREADTHROUGHENTIREPROCEDUREBEFOREBEGINNING!

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