Background:Lysozyme(LZ)isacationicantimicrobialpeptideof14kDa.Lysozymeisstoredinprimarybutpredominantlyinspecific(secondary)granulesofneutrophils.ItcleavespeptidoglycanconstituentsofthebacterialcellwallandcanbindLPS.TheepitoperecognizedbyantibodyLZ-2isexpressedbyvirtuallyallmyeloidcellsincludingnormalandmalignantgranulocytesandmonocytes.InnormalmyelopoiesisLZcanfirstbedetectedatthemyeloblaststagewhereitappearssomewhatlaterthanMPOexpression.Lactoferrin(LF)isaniron-bindingproteinwithbactericidalandbacteriostaticactivity,whichisstoredwithinthesecondarygranulesofgranulocytes.LFexpressionisrestrictedtothepost-mitoticmaturationcompartmentofthegranulocyticlineage,startingfromthemyelocytestage.NormalandmalignantmyeloblastsareLFnegative.ThecombinedstainingforLysozymeandLactoferrinallowsthedistinctionbetweenmatureandimmaturemyelomonocyticcells.TheLZ/LFCOMBI-ICreagentpermitstheidentificationandenumerationofmyeloidcellsinnormalandmalignanthumanbloodandbonemarrowsamplesusingflowcytometry.Resultsmustbeputwithinthecontextofotherdiagnostictestsaswellastheclinicalhistoryofthepatientbyacertifiedprofessionalbeforefinalinterpretation.Analysesperformedwiththisantibodyshouldbeparalleledbypositiveandnegativecontrols.Ifunexpectedresultsareobtained,whichcannotbeattributedtodifferencesinlaboratoryprocedures,pleasecontactusProduct:1mlofFITC-conjugatedantiLysozyme(cloneLZ-2)andPE-conjugatedantiLactoferrin(clone4C5)inPBSpH7.2,1%BSA,and0.05%NaN3,approximately50tests.Applications:PermeABIlizationandStainingProcedure-IncombinationwithourPermeabilizationKitFIX&PERM(Cat.No.GAS-002)intracellularLysozymeandLactoferrincanbeeasilystainedincellsUSPensions.-Foreachsampletobeanalyzedadd50µlofwholeblood,bonemarrowormononuclearcellsuspensionina5mltube-Add100µlofReagentA(FixationMedium,storedandusedatroomtemperature)-Incubatefor15minutesatroomtemperature-Add5mlphosphatebufferedsalineandcentrifugecellsfor5minutesat300g-Removesupernatantandaddtocellpellet100µlReagentB(PermeabilizationMedium)and20µloftheLZ/LFCOMBI-ICmonoclonalantibodyconjugate-Vortexatlowspeedfor1-2seconds-Incubatefor15minutesatroomtemperature-Washcellswithphosphatebufferedsalineasdescribedabove-Removesupernatantandresuspendcellsinsheathfluidforimmediateanalysisorresuspendcellsin0.5ml1.0%formaldehydeandstorethemat2-8°Cinthedark.Analyzefixedcellswithin24hours.Specificity:Theanti-LysozymeAntibody(cloneLZ-2)reactswithintracellularhumanlysozyme/muramidaseexpressedbyvirtuallyallmyelomonocyticcells,macrophagesandtheirprecursors.TheLFmAb(clone4C5)recognizeslactoferrinstoredwithinsecondarygranulesofpostmitoticgranulocyte-commitedcells.InthisCOMBI-ICReagentantibodyLZ-2isconjugatedtoFITC,antibody4C5isconjugatedtoPhycoeythrin(PE).ThesensitivityyofLZ/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:1.Knapp,W.,Majdic,O.&Strobl,H.(1993)RecentResultsCancerRes131,31-40.2.Konikova,E.,Glasova,M.,Kusenda,J.&Babusikova,O.(1998)Neoplasma45,282-91.3.Lanza,F.,Latorraca,A.,Moretti,S.,Castagnari,B.,Ferrari,L.&Castoldi,G.(1997)Cytometry30,134-44.4.Oehler,L.,Majdic,O.,Pickl,W.F.,Stockl,J.,Riedl,E.,Drach,J.,Rappersberger,K.,Geissler,K.&Knapp,W.(1998)JExpMed187,1019-28.5.Paietta,E.(2003)BestPractResClinHaematol16,671-83.6.Rado,T.A.,Bollekens,J.,StLaurent,G.,Parker,L.&Benz,E.J.,Jr.(1984)Blood64,1103-9.7.Srivastava,C.H.,Rado,T.A.,Bauerle,D.&Broxmeyer,H.E.(1991)JImmunol146,1014-9.8.Strobl,H.&Knapp,W.(2004)JBiolRegulHomeostAgents18,335-9.9.Teng,C.T.,Gladwell,W.,Beard,C.,Walmer,D.,Teng,C.S.&Brenner,R.(2002)MolHumReprod8,58-67.10.Braylan,R.C.,Orfao,A.,Borowitz,M.J.&Davis,B.H.(2001)Cytometry46,23-7.11.Catovsky,D.,Matutes,E.,Buccheri,V.,Shetty,V.,Hanslip,J.,Yoshida,N.&Morilla,R.(1991)AnnHematol62,16-21.Caution:Forprofessionalusersonly.Thisreagentcontainssodiumazide.Toavoidthedevelopmentofhazardousconditions,reagentscontainingazideshouldbedilutedinrunningwaterpriortobediscarded.SimilartotheworkwithotherBIOLOGicalproducts,properhandlingproceduresarerecommended.