Nordicmubio/IgG1 Negative Control/GM-4991

Background:

ThisreadytouseNegativeControlreagentcontainspurified,fluoresceinorphycoerythrinconjugatedmouseimmunoglobulinmoleculesofIgG1isotype,whichhavebeenselectedonthebasisoftheirbindingcharacteristics:nospecificbindingtohumancellsurfaceorintracellularantigens,samelowrangeofnonspecificbindingtohumanleukocytesasotherNordic-MubioReagents.ThisisotypecontrolIgG1issuitableasaNegativeControltobeusedincombinationwithNordic-MUbioreagentsforthe:-EnumerationofMyeloidCells-AnalysisofMyeloidDifferentiationStage-EnumerationofB-cellsandPrecursors-EnumerationofT-cellsandPrecursors-AnalysisofLeukemiaCells-AnalysisofImmunodeficiencyStatesThenegativecontrolreagentpermitstoestimatethedegreeofnon-specificbindingofisotypematchedimmunologbulinstoleukocytesviae.g.Fc-receptors.Itenablestheexperttosetflowcytometricparametersaccordingly.Resultsmustbeputwithinthecontextofotherdiagnostictestsaswellastheclinicalhistoryofthepatientbyacertifiedprofessionalbeforefinalinterpretation.

Product:

2mlofnon-conjugatedVI-APinPBSpH7.2,1%BSA,and0.05%NaN3,approximately100tests.

Applications:

DirectImmunofluorescence(StainingProcedure)Nordic-MUbioFluorochromelabeledantibodiesaredesignedforusewitheitherwholebloodorisolatedmononuclearcell(MNC)preparations.Proposedstainingprocedureforwholebloodinshort:-Foreachsampleadd50µlofEDTAanti-coagulatedbloodtoa3-5mltube-Add20µloftheappropriateNordic-MUbiomonoclonalantibodyconjugate-Incubatethetubefor15minutesat4°Coratroomtemperatureinthedark-Add100µlNM-LYSE(Cat.No.GAS-003)toeachtubeandincubatefor10minutesatroomtemperature-Add3-4mlofdestilledwaterandvortex,incubatefor5-10minutesatroomtemperature-Centrifugetubefor5minutesat300g-AspiratesupernatantandresUSPendpelletin0.3mlofsheathfluid-Analyzeimmediatelyorstoresamplesat2-8°Cinthedarkandanalyzewithin24hoursFor“No-Wash”protocolpleaserefertowww.Nordicmubio.comProposedstainingprocedureforMNCinshort:-Carefullyadd20µlantibodyconjugateand50-100µlMNCtothebottomofatube-Vortexatlowspeedfor1-2seconds-Incubatefor15-30minutesat2-8°Coratroomtemperature-Centrifugetubesfor5minutesat300g-Removesupernatant,resuspendcellsin2-5mlofphosphatebufferedsaline(PBS)andcentrifugecellsagainfor5minutesat300g-Removesupernatantandresuspendcellsinsheathfluidforimmediateanalysisorresuspendcellsin0.5ml1%formaldehydeandstorethemat2-8°Cinthedark.Analyzefixedcellswithin24hours

Specificity:

ThecloneVI-APreactswithcalfintestinealkalinephosphataseanddoesnotshowcross-reactivitywithhumanproteins.ThesensitivityofVI-APmAbisdeterminedbystainingwell-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.

Caution:

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