| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 360/449 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection HydrolyticEnzymes |
| Related | DiagnosticMolecules BiochemicalAssays |
1.Prepareneuraminidasestandardstocksolution:
Add50µLofddH2OintothevialofNeuraminidaseStandard(ComponentC)tomake2U/mLneuraminidasestandardstocksolution.
Note:Theconcentrationofthisstocksolutionisapproximately2U/mL.TheunusedNeuraminidaseStandardsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.Prepare200XFluLiteTMBluestocksolution:
Add50µLofddH2OintothevialofFluLiteTMBlue(ComponentA)tomake200Xstocksolution.
Note:TheunusedFluLiteTMBluesolutionshouldbedividedintosingleusealiquotsandstoredat-20oCandkeptfromlight.
3.Prepareneuraminidaseassaymixture:
Add25µLof200XFluLiteTMBluestocksolution(fromStep2)into5mLofAssayBuffer(ComponentB),andmixwell.
4.Prepareserialdilutionsofneuraminidasestandard(0to20mU/mL):
4.1 Add10μLof2U/mLneuraminidasestandardstocksolution(fromStep1)to990µLofassaybuffer(ComponentB)togenerate20mU/mLneuraminidasestandard.
Note:Dilutedneuraminidasestandardsolutionisunstable.Usewithin4hours.
4.2 Take500μLof20mU/mLneuraminidasestandardsolution(fromStep4.1)toperform1:2serialdilutionstoget10,5,2.5,1.25,0.625,0.312and0mU/mLserialdilutionsofneuraminidasestandard.
4.3 Addneuraminidasestandardsandneuraminidase-containingtestsamplesintoasolidblack96-wellmicroplateasshowninTables1and2.
Table1Layoutofneuraminidasestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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NA1 | NA1 | …. | …. | …. | …. |
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NA2 | NA2 |
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NA3 | NA3 |
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NA4 | NA4 |
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NA5 | NA5 |
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NA6 | NA6 |
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NA7 | NA7 |
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Note:NA=NAStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
NAStandard | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer:50μL | 50μL |
*Note:Addtheserialdilutionsofneuraminidasestandardfrom0.312mUto20mUintowellsfromNA1toNA7induplicate.
5.Runneuraminidaseassay:
5.1 Add50μLofneuraminidaseassaymixture(fromStep3)toeachwelloftheneuraminidasestandard,blankcontrol,andtestsamples(seeStep4.3)tomakethetotalneuraminidaseassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofneuraminidasereactionmixtureintoeachwell.
5.2 Incubatethereactionat37oCorroomtemperaturefor1to2hours,protectedfromlight.
Note:37oCincubationgivesbetterresults.
5.3 MonitorthefluorescenceincreaseatEx/Em=320/460nm(cutoff=420nm)withafluorescencemicroplatereader.
| References&Citations |  PrinterFriendlyVersion |
- SandbulteMR,GaugerPC,KitikoonP,ChenH,PerezDR,RothJA,VincentAL.(2016)NeuraminidaseinhibitingantibodyresponsesinpigsdifferbetweeninfluenzaAvirusN2lineagesandbyvaccinetype.Vaccine,34,3773.
- ZhangM,ZhaoH,ZhaoZ,YanH,LvR,CuiL,YuanJ,WangD,GengY,LiuD,WangX.(2016)Rapidscreening,identification,andpurificationofneuraminidaseinhibitorsfromLithospermumerythrorhizonSieb.etZucc.byultrafiltrationwithHPLC-ESI-TOF-MScombinedwithsemipreparativeHPLC.JSepSci,39,2097.
- HuangW,TanM,ZhaoX,ChengY,LiX,GuoJ,WeiH,XiaoN,WangZ,WangD,ShuY.(2015)[SusceptibilityofhumaninfluenzaA(H3N2)virusestoneuraminidaseinhibitorsisolatedduring2011-2012inChina].ZhonghuaYuFangYiXueZaZhi,49,481.
- SomasundaramB,FeeCJ,FredericksR,WatsonAJ,FairbanksAJ.(2015)Developmentofasurfaceplasmonresonanceassaytomeasurethebindingaffinityofwild-typeinfluenzaneuraminidaseanditsH274Ymutanttotheantiviraldrugzanamivir.JMolRecognit,28,87.
- PedersenJC.(2014)Neuraminidase-inhibitionassayfortheidentificationofinfluenzaAvirusneuraminidasevirussubtypeorneuraminidaseantibodyspecificity.MethodsMolBiol,1161,27.
- SmolonoginaTA,DeshevaIuA,RekstinAR,MironovAN,RudenkoLG.(2013)[Evaluationoftheanti-neuraminidaseantibodiesinclinicaltrialsoftheliveinfluenzavaccineoftheA(H5N2)subtype].VoprVirusol,58,31.
- LuX,LiuF,ZengH,SheuT,AchenbachJE,VeguillaV,GubarevaLV,GartenR,SmithC,YangH,StevensJ,XuX,KatzJM,TumpeyTM.(2014)Evaluationoftheantigenicrelatednessandcross-protectiveimmunityoftheneuraminidasebetweenhumaninfluenzaA(H1N1)virusandhighlypathogenicavianinfluenzaA(H5N1)virus.Virology,454-455,169.
- ZhangY,FuD,ChenH,ZhangZ,ShiQ,ElsayedAK,LiB.(2013)PartialantiviralactivitiesdetectionofchickenMxjointingwithneuraminidasegene(NA)againstNewcastlediseasevirus.PLoSOne,8,e71688.
- ChoiKS,KyeSJ,JeonWJ,ParkMJ,KimS,SeulHJ,KwonJH.(2013)Preparationanddiagnosticutilityofahemagglutinationinhibitiontestantigenderivedfromthebaculovirus-expressedhemagglutinin-neuraminidaseproteingeneofNewcastlediseasevirus.JVetSci,14,291.
- HurtAC,Okomo-AdhiamboM,GubarevaLV.(2012)Thefluorescenceneuraminidaseinhibitionassay:afunctionalmethodfordetectionofinfluenzavirusresistancetotheneuraminidaseinhibitors.MethodsMolBiol,865,115.
