| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy ReactiveOxygenSpecies |
| Related | RedoxEnzymes BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1 Amplite™Redstocksolution(250X):Add40µLofDMSO(ComponentE)intothevialofAmplite™Redsubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandfrozenat-20°C.
Note1:Avoidrepeatedfreeze-thawcycles.
Note2:TheAmplite™Redsubstrateisunstableinthepresenceofthiolssuchasdithiothreitol(DTT)and2-mercaptoethanol.ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbenohigherthan10μM.TheAmplite™RedsubstrateisalsounstableathighpH(>8.5).Therefore,thereactionshouldbeperformedatpH7–8.Theprovidedassaybuffer,pH7.4,isrecommended.
1.2 10mMH2O2stocksolution(500X):Add10µLof3%H2O2(0.88M,ComponentC)into870µLofAssayBuffer(ComponentB).
Note:ThedilutedH2O2solutionisnotstable.Theunusedportionshouldbediscarded.
1.3 200mU/mLmyeloperoxidasestocksolution:Add50µLofAssayBuffer(ComponentB)intothevialofMyeloperoxidaseStandard(ComponentD).
Note:Onevialcontainsapproximately5~10mUmyeloperoxidase.TheunusedMPOstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20°C.
2.Prepareassaymixture:
PrepareassaymixtureaccordingtoTable1andprotectfromlight.
Table1.Assaymixtureforone96-wellplate
Components | Volume |
Amplite™RedStockSolution(250X,fromStep1.1) | 20µL |
H2O2(500X,fromStep1.2) | 10µL |
AssayBuffer(ComponentB) | 5mL |
Totalvolume | 5.03mL |
3.PrepareseriallydilutedMPOstandards(0to10mU/mL):
3.1 Add20μLof200mU/mLMPOstocksolution(fromStep1.3)into380μLofAssayBuffer(ComponentB)toget10mU/mLMPOstandardsolution.
3.2 Take150μLof10mU/mLMPOstandardsolutiontoperform1:3serialdilutionstoget3,1,0.3,0.1,0.03,0.01and0mU/mLseriallydilutedMPOstandards.
3.3 AddMPOstandardsand/orMPO-containingtestsamplesintoa96-wellsolidblackmicroplateasdescribedinTables2and3
Table2.LayoutofMPOstandardsandtestsamplesina96-wellsolidblackmicroplate
BL | BL | TS | TS | …. | …. |
|
|
|
|
|
|
MPO1 | MPO1 | …. | …. | …. | …. |
|
|
|
|
|
|
MPO2 | MPO2 |
|
|
|
|
|
|
|
|
|
|
MPO3 | MPO3 |
|
|
|
|
|
|
|
|
|
|
MPO4 | MPO4 |
|
|
|
|
|
|
|
|
|
|
MPO5 | MPO5 |
|
|
|
|
|
|
|
|
|
|
MPO6 | MPO6 |
|
|
|
|
|
|
|
|
|
|
MPO7 | MPO7 |
|
|
|
|
|
|
|
|
|
|
Note:MPO=myeloperoxidasestandards,BL=blankcontrol,TS=testsamples.
Table3.Reagentcompositionforeachwell
MPOStandard | BlankControl | TestSample |
SerialDilutions*(50μL) | AssayBuffer(ComponentB):50μL | 50μL |
*Note1:Addtheseriallydilutedmyeloperoxidasestandardsfrom0.01mU/mLto10mU/mLintoeachwellfromMPO1toMPO7induplicate.
Note2:HighconcentrationofMPOmaycausereducedfluorescencesignalduetotheoveroxidationofAmplite™Redsubstrate(toanon-fluorescentproduct).
4.RunMPOassay:
4.1 Add50μLofassaymixture(fromStep2)intoeachwelloftheMPOstandards,blankcontrol,andtestsamples(seeStep3,Table2)tomakethetotalMPOassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofassaymixtureintoeachwell.
4.2 Incubatethereactionfor30to60minutesatroomtemperature,protectedfromlight.
4.3 MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=530-570nm/590-600nm(optimalEx/Em=540/590nm,cutoff=570nm).
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.TheabsorptiondetectionhaslowersensitivitycomparedtofluorescencereADIng.
| References&Citations |  PrinterFriendlyVersion |
1. TanS,WangG,PengM,ZhangX,ShenG,JiangJ,ChenF.(2009)Detectionofmyeloperoxidaseactivityinprimaryleukemiccellsbyanenhancedchemiluminescentassayfordifferentiationbetweenacutelymphoblasticandnon-lymphoblasticleukemia.ClinChimActa,403,216.
2. ZelzerS,KhoschsorurG,StettinM,WeihrauchG,Truschnig-WildersM.(2009)DeterminationofmyeloperoxidaseinEDTAplasma:comparisonofanenzyme-linkedimmunosorbentassaywithachemiluminescentautomatedimmunoassay.ClinChimActa,406,62.
3. FietzS,BondzioA,MoschosA,HertschB,EinspanierR.(2008)Measurementofequinemyeloperoxidase(MPO)activityinsynovialfluidbyamodifiedMPOassayandevaluationofjointdiseases-aninitialcasestudy.ResVetSci,84,347.
4. GrulkeS,FranckT,GanglM,PetersF,SalcicciaA,Deby-DupontG,SerteynD.(2008)Myeloperoxidaseassayinplasmaandperitonealfluidofhorseswithgastrointestinaldisease.CanJVetRes,72,37.
5. SakamotoW,FujiiY,KanehiraT,AsanoK,IzumiH.(2008)Anovelassaysystemformyeloperoxidaseactivityinwholesaliva.ClinBiochem,41,584.
6. DypbuktJM,BishopC,BrooksWM,ThongB,ErikssonH,KettleAJ.(2005)Asensitiveandselectiveassayforchloramineproductionbymyeloperoxidase.FreeRadicBiolMed,39,1468.
7. FranckT,GrulkeS,Deby-DupontG,DebyC,DuvivierH,PetersF,SerteynD.(2005)Developmentofanenzyme-linkedimmunosorbentassayforspecificequineneutrophilmyeloperoxidasemeasurementinblood.JVetDiagnInvest,17,412.
8. KapuscinskaR,WysockaJ,NiczyporukW,RatomskiK.(2004)[CytofluorimetricassayforevaluationofCD16receptorexpressionandmyeloperoxidase(MPO)activityofneutrophilsinpatientswithpsoriasisvulgaristreatedwithPUVA].WiadLek,57,599.
9. YuF,ZhaoMH,ZhangYK,WangHY.(2003)[Therelationshipbetweensubclassificationofanti-myeloperoxidaseIgGbyenzyme-linkedimmuno-sorbentassayanalysisandvasculitisactivity].ZhonghuaNeiKeZaZhi,42,27.
10. HaqqaniAS,SandhuJK,BirnboimHC.(1999)Amyeloperoxidase-specificassaybaseduponbromide-dependentchemiluminescenceofluminol.AnalBiochem,273,126.
