AntibodiesOnline/Myostatin (MSTN) ELISA Kit/ABIN1081774/96 tests

ThisassayhashighsensitivityandexcellentspecificityfordetectionofMyostatin(MSTN).
Nosignificantcross-reactivityorinterferencebetweenMyostatin(MSTN)andanalogueswasobserved.

• Pre-coated,readytouse96-wellstripplate
• Platesealerfor96wells
• StandardDiluent
• AssayDiluentA
• AssayDiluentB
• StopSolution
• Standard
• DetectionReagentA
• DetectionReagentB
• TMBSubstrate
• WashBuffer(30×concentrate)
• Instructionmanual

• Luminometercapableofreading96-wellmicroplateswiththefollowingparameters:lagtime30.0secs,readtime1.0sec/well.
• Precisionsingleormulti-channelpipettesandpipettetipswithdisposabletips.
• EppendorfTubesfordilutingsamples.
• Deionizedordistilledwater.
• Absorbentpaperforblottingthemicrotiterplate.
• ContainerforWashSolution

• Limitedbythecurrentconditionandscientifictechnology,wecannotcompletelyconductthecomprehensiveidentificationandanalysisontherawmaterialprovidedbysuppliers.Sotheremightbesomequalitativeandtechnicalriskstousethekit.
• Thefinalexperimentalresultswillbecloselyrelatedtovalidityoftheproducts,operationskillsoftheendusersandtheexperimentalenvironments.Pleasemakesurethatsufficientsamplesareavailable.
• Kitsfromdifferentbatchesmaybealittledifferentindetectionrange,sensitivityandcolordevelopingtime.
• Donotmixorsubstitutereagentsfromonekitlottoanother.Useonlythereagentssuppliedbymanufacturer.
• Protectallreagentsfromstronglightduringstorageandincubation.Allthebottlecapsofreagentsshouldbecoveredtightlytopreventtheevaporationandcontaminationofmicroorganism.
• Theremaybesomefoggysubstanceinthewellswhentheplateisopenedatthefirsttime.Itwillnothaveanyeffectonthefinalassayresults.Donotremovemicrotiterplatefromthestoragebaguntilneeded.
• Wrongoperationsduringthereagentspreparationandloading,aswellasincorrectparametersettingfortheplatereadermayleadtoincorrectresults.Amicroplateplatereaderwithabandwidthof10nmorlessandanopticaldensityrangeof0-3O.D.orgreaterat450±10nmwavelengthisacceptableforuseinabsorbancemeasurement.Pleasereadtheinstructioncarefullyandadjusttheinstrumentpriortotheexperiment.
• Eventhesameoperatormightgetdifferentresultsintwoseparateexperiments.Inordertogetbetterreproducibleresults,theoperationofeverystepintheassayshouldbecontrolled.Furthermore,apreliminaryexperimentbeforeassayforeachbatchisrecommended.
• EachkithasbeenstrictlypassedQ.Ctest.However,resultsfromendusersmightbeinconsistentwithourin-housedataduetosomeunexpectedtransportationconditionsordifferentlabequipments.Intra-assayvarianceamongkitsfromdifferentbatchesmightarisefromabovefactors,too.
• Kitsfromdifferentmanufacturersforthesameitemmightproducedifferentresults,sincewehavenotcomparedourproductswithothermanufacturers.

Informationonstandardmaterial:
Thestandardmightberecombinantproteinornaturalprotein,thatwilldependonthespecifickit.Moreover,theexpressionsystemisE.colioryeastormammalcell.Thereis0.05%proclin300inthestandardaspreservative.

Informationonreagents:
Thestopsolutionusedinthekitissulfuricacidwithconcentrationof1mol/L.AndthewashsolutionisTBS.Thestandarddiluentcontains0.02%sodiumazide,assaydiluentAandassaydiluentBcontain0.01%sodiumazide.SomekitscancontainisBSAinthem.

Informationonantibodies:
Theprovidedantibodiesandtheirhostvaryindifferentkits.

• Bringallkitcomponentsandsamplestoroomtemperature(18-25°C)beforeuse.
• Standard-ReconstitutetheStandardwith1.0mLofStandardDiluent,keptfor10minutesatroomtemperature,shakegently(nottofoam).Theconcentrationofthestandardinthestocksolutionis50ng/mL.Pleaseprepare7tubescontaining0.5mLStandardDiluentandproduceadoubledilutionseries.Mixeachtubethoroughlybeforethenexttransfer.Setup7pointsofdilutedstandardsuchas50ng/mL,25ng/mL,12.5ng/mL,6.25ng/mL,3.12ng/mL,1.56ng/mL,0.78ng/mL,andthelastEPtubeswithStandardDiluentistheblankas0ng/mL.
• AssayDiluentAandAssayDiluentB-Dilute6mLofAssayDiluentAorBConcentrate(2×)with6mLofdeionizedordistilledwatertoprepare12mLofAssayDiluentAorB.Thepreparedworkingdilutioncannotbefrozen.(Infact,morethan6mLAssayDiluentAandAssayDiluentBarecontainedinthebottles.Therefore,ineverytest,pleasepreciselypipetterequiredamountofDiluentandmakedoubledilutioninanewcontainer.Thepreparedworkingdilutioncannotbefrozen.)
• DetectionReagentAandDetectionReagentB-BrieflyspinorcentrifugethestockDetectionAandDetectionBbeforeuse.DilutetotheworkingconcentrationwithworkingAssayDiluentAorB,respectively(1:100).
• WashSolution-Dilute20mLofWashSolutionconcentrate(30×)with580mLofdeionizedordistilledwatertoprepare600mLofWashSolution(1×).
• SubstrateworkingSolution-MixthesubstrateAandBbytheratioof99:1tomakethesubstrateworkingsolution.Mixthoroughly.Forexample,prepare1,000µLSubstrateworkingSolutionwith990µLSubstrateA+10µLSubstrateB.

• Makingserialdilutioninthewellsdirectlyisnotpermitted.
• Preparestandardwithin15minutesbeforeassay.Pleasedonotdissolvethereagentsat37°Cdirectly.
• PleasecarefullyreconstituteStandardsorworkingDetectionReagentAandBaccordingtotheinstruction,andavoidfoamingandmixgentlyuntilthecrystalsarecompletelydissolved.Tominimizeimprecisioncausedbypipetting,usesmallvolumesandensurethatpipettorsarecalibrated.Itisrecommendedtosuckmorethan10µLforoncepipetting.
• ThereconstitutedStandards,DetectionReagentAandDetectionReagentBcanbeusedonlyonce.
• PrepareSubstrateworkingSolutionwithin15minutesbeforeassay.
• IfcrystalshaveformedintheWashSolutionconcentrate(30×),warmtoroomtemperatureandmixgentlyuntilthecrystalsarecompletelydissolved.
• Contaminatedwaterorcontainerforreagentpreparationwillinfluencethedetectionresult.

• Weareonlyresponsibleforthekititself,butnotforthesamplesconsumedduringtheassay.Theusershouldcalculatethepossibleamountofthesamplesusedinthewholetest.Pleasereservesufficientsamplesinadvance.
• Pleasepredicttheconcentrationbeforeassaying.Ifvaluesforthesearenotwithintherangeofthestandardcurve,usersmustdeterminetheoptimalsampledilutionsfortheirparticularexperiments.Sampleshouldbedilutedby0.01mol/LPBS(PH=7.0-7.2).
• Ifthesamplesarenotindicatedinthemanual,apreliminaryexperimenttodeterminethevalidityofthekitisnecessary.
• TissueorcellextractionsamplespreparedbychemicallysisbuffermaycauseunexpectedELISAresultsduetotheimpactsfromcertainchemicals.
• Duetothepossibilityofmismatchingbetweenantigenfromotheroriginandantibodyusedinourkits(e.g.antibodytargetsconformationalepitoperatherthanlinearepitope),somenativeorrecombinantproteinsfromothermanufacturersmaynotberecognizedbyourproducts.
• Influencedbythefactorsincludingcellviability,cellnumberorsamplingtime,samplesfromcellculturesupernatantmaynotbedetectedbythekit.
• Freshsampleswithoutlongtimestorageisrecommendedforthetest.Otherwise,proteindegradationanddenaturalizationmayoccurinthosesamplesandfinallyleadtowrongresults.

1. Prepareallreagents,samplesandstandards,
   
2. Add100μLstandardorsampletoeachwell.Incubate2hoursat37 °C,
   
3. Aspirateandadd100μLpreparedDetectionReagentA.Incubate1hourat37 °C,
   
4. Aspirateandwash3times,
   
5. Add100μLpreparedDetectionReagentB.Incubate30 minutesat37 °C,
   
6. Aspirateandwash5times,
   
7. Add100μLSubstrateSolution.Incubate10 minutesat37 °C,
   
8. ReadRLUvalueimmediately.

Averagetheduplicatereadingsforeachstandard,control,andsamplesandsubtracttheaveragezerostandardrelativelightunit(RLU).Createastandardcurveonlog-loggraphpaper,withMSTNconcentrationonthey-axisandtheRLUvalueonthex-axis.Drawthebestfitstraightlinethroughthestandardpointsanditcanbedeterminedbyregressionanalysis.Usingsomeplotsoftware,suchascurveexpert1.30,isalsorecommended.Ifsampleshavebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.
Inordertomakethecalculationeasier,weplottheRLUvalueofthestandard(X-axis)againsttheknownconcentrationofthestandard(Y-axis),althoughconcentrationisindeedtheindependentvariablewhileRLUvalueisthedependentvariable.Further,inthispart,inordertohelpthecustomerperformtheassaymorevisual,weprovidethecustomerwiththerawdata(notthelogofdata).However,plottinglogofthedatatoconstructthecurvewillberecommended.TheRLUvaluesofthestandardcurvemayvaryaccordingtotheconditionsofassayperformance(e.g.operator,pipettingtechnique,washingtechniqueortemperatureeffects).Thiscurveisprovidedfordemonstrationonly.Thecustomersshouldestablishtheirownstandardcurveforeachtestconducted.

Intra-assayPrecision(Precisionwithinanassay):3sampleswithlow,middleandhighlevelMyostatin(MSTN)weretested20timesononeplate,respectively.
Inter-assayPrecision(Precisionbetweenassays):3sampleswithlow,middleandhighlevelMyostatin(MSTN)weretestedon3differentplates,8replicatesineachplate.
CV(%)=SD/meanX100
Intra-Assay:CV<10%
Inter-Assay:CV<12%

Thestabilityofkitisdeterminedbythelossrateofactivity.Thelossrateofthiskitislessthan5 %withintheexpirationdateunderappropriatestoragecondition.
Tominimizeextrainfluenceontheperformance,operationproceduresandlabconditions,especiallyroomtemperature,airhumidity,incubatortemperatureshouldbestrictlycontrolled.Itisalsostronglysuggestedthatthewholeassayisperformedbythesameoperatorfromthebeginningtotheend.

• Forunopenedkit:Allthereagentsshouldbekeptaccordingtothelabelsonvials.TheStandard,DetectionReagentA,DetectionReagentBandthe96-wellstripplateshouldbestoredat-20°Cuponreceiptwhiletheothersshouldbeat4°C.
• Foropenedkit:Whenthekitisopened,theremainingreagentsstillneedtobestoredaccordingtotheabovestoragecondition.Besides,pleasereturntheunusedwellstothefoilpouchcontainingthedesiccantpack,andresealalongentireedgeofzip-seal.
  Note:Itishighlyrecommendedtousetheremainingreagentswithin1monthprovidedthisiswithintheexpirationdateofthekit.
• ForELISAkit,1daystorageat37°Ccanbeconsideredas2monthsat4°C,whichmeans3daysat37°Cequaling6monthsat4°C.