Background:

Myeloperoxidase(MPO)isaglycoproteinpresentintheazurophil(primary)granulesofmyeloidcells,whichappearsinthemyeloblaststageofmyeloidcelldifferentiation.MPOishemostcommonfunctionalproteinofmyeloidcellsandisinvolvedintheinflammatoryresponse.Ithelpstokillmicrobesbybreakingdownperoxideinthepresenceofhalideions,contributingtothebactericidalfunctionofgranulocytes.TheprimarytranslationproductofMPOundergoesglycosylationwithproductionofthe89kDaheme-freeapopro-MPOformfollowedbyincorporationofhemeandconversionintotheenzymaticallyactivepro-MPOform.Subsequently,pro-MPObecomestargetedtoazurophilgranuleswherefinalprocessingoccurstoproducematuredimericMPOconsistingofthe59-64kDaMPOα-chainandthe14kDaMPOβ-chain.Lactoferrin(LF)isaniron-bindingproteinwithbactericidalandbacteriostaticactivitywhichisstoredwithinthesecondarygranulesofgranulocytes.LFexpressionisrestrictedtothepost-mitoticmaturationcompartmentofthegranulocyticlineage,startingfromthemyelocytestage.NormalandmalignantmyeloblastsareLFnegative.ThecombinedstainingforMPOandLFallowsthedistinctionbetweenmatureandimmaturemyelomonocyticcells.TheMPO-C2/LFCOMBI-ICreagentpermitstheidentificationandenumerationofimmatureandmorematuremyelomonocyticcellpopulationsinnormalandmalignanthumanbloodandbonemarrowusingflowcytometry.Resultsmustbeputwithinthecontextofotherdiagnostictestsaswellastheclinicalhistoryofthepatientbyacertifiedprofessionalbeforefinalinterpretation.Analysesperformedwiththisantibodyshouldbeparalleledbypositiveandnegativecontrols.Ifunexpectedresultsareobtainedwhichcannotbeattributedtodifferencesinlaboratoryprocedures,pleasecontactus.

Product:

1mlofFITC-conjugatedantiMyeloperoxidase-C2(clone8E6)andPE-conjugatedantiLactoferrin(clone4C5)inPBSpH7.2,1%BSA,and0.05%NaN3,approximately50tests.

Applications:

PermeABIlizationandStainingProcedure-IncombinationwithourPermeabilizationKitFIX&PERM(Cat.No.GAS-002)intracellularMPO-C2andLFcanbeeasilystainedincellsUSPensions.-Foreachsampletobeanalyzedadd50µlofwholeblood,bonemarrowormononuclearcellsuspensionina5mltube-Add100µlofReagentA(FixationMedium,storedandusedatroomtemperature)-Incubatefor15minutesatroomtemperature-Add5mlphosphatebufferedsalineandcentrifugecellsfor5minutesat300g-Removesupernatantandaddtocellpellet100µlReagentB(PermeabilizationMedium)and20µloftheMPO-C2/LFCOMBI-ICmonoclonalantibodyconjugate-Vortexatlowspeedfor1-2seconds-Incubatefor15minutesatroomtemperature-Washcellswithphosphatebufferedsalineasdescribedabove-Removesupernatantandresuspendcellsinsheathfluidforimmediateanalysisorresuspendcellsin0.5ml1.0%formaldehydeandstorethemat2-8°Cinthedark.Analyzefixedcellswithin24hours

Specificity:

AntibodyMPO-C2(clone8E6)reactswithhumanmyeloperoxidase(MPO)expressedbynormalandmalignantmyelomonocyticcells.TheLFmAb(clone4C5)recognizeslactoferrinstoredwithinsecondarygranulesofpostmitoticgranulocyte-commitedcells.InthisCOMBI-ICReagentantibody8E6isconjugatedtoFITC,antibody4C5isconjugatedtoPhycoeythrin(PE).ThesensitivityofMPO-C2/LFmAbisdeterminedbystainingwell-definedbloodsamplesfromrepresentativedonorswithserial-foldmAbdilutionstoobtainatitrationcurvethatallowsrelatingthemAbconcentrationtothepercentageofstainedcellsandgeometricMFI(meanfluorescenceintensity).Forthispurpose,amAb-concentrationrangeisselectedtoincludeboththesaturationpoint(i.e.themAbdilutionexpectedtobindallepitopesonthetargetcell)andthedetectionthreshold(i.e.themAbdilutionexpectedtorepresenttheleastamountofmAbneededtodetectanidenticalpercentageofcells).Inpractice,50µlofleukocytescontaining10^7cells/mlarestainedwith20µlmAbofvariousdilutionstoobtainatitrationcurveandtoidentifythesaturationpointanddetectionthreshold.Thefinalconcentrationoftheproductisthenadjustedtobeatleast3-foldabovethedetectionthreshold.Inadditionandtocontrollot-to-lotvariation,thegivenlotiscomparedandadjustedtofluorescencestandardswithdefinedintensity.

Storage:

Nordic-Mubiomonoclonalantibodyreagentscontainoptimalconcentrationsofaffinity-purifiedantibody.Forstabilityreasonsthismonoclonalantibodysolutioncontainssodiumazide.Thesereagentsshouldbestoredat2-8°C(DONOTFREEZE!)andprotectedfromprolongedexposuretolight.Stabilityofthereagent:Pleaserefertotheexpirydateprintedontothevial.Theuseofthereagentaftertheexpirationdateisnotrecommended.

References:

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Caution:

Forprofessionalusersonly.Thisreagentcontainssodiumazide.Toavoidthedevelopmentofhazardousconditions,reagentscontainingazideshouldbedilutedinrunningwaterpriortobediscarded.SimilartotheworkwithotherBIOLOGicalproducts,properhandlingproceduresarerecommended.

Noridic-Mubio是荷兰的一家著名的免疫学试剂生产厂商，因收购An Der Grub Bio Research GmbH(ADG)，增加了自己的产品线：全球最好的固定破膜剂FIX&PERM 。Nordic-Mubio严格遵照ISO9001标准生产高质量的抗体，主要为针对细胞粘附蛋白、核蛋白和骨架蛋白的抗体。同时Nordic-Mubio也是斑马鱼抗体开发的引领者之一，拥有50多种可用于斑马鱼免疫荧光检测的抗体。