MouseMannmabindingprotein/mannanbindinglectin,MBP/MBLELISAkitCatalogNo.HR-M0373(96tests)FORRESEARCHUSEONLYIntendeduseThisimmunoassaykitallowsfortheuseinvitroquantitativedeterminationofmouseMannmabindingprotein/mannanbindinglectin,MBP/MBLconcentrati***incellculturesupernates,serum,pla***aandotherbiologicalfluids.IntroductionMannose(ormannan)-bindinglectin(MBL),alsoknownasserummannosebindingprotein(MBP),initiatesthelectinbranchoftheinnateimmuneresp***ebybindingtothesurfaceofpotentiallypathogenicmicroorgani***sandinitiatingcomplementfixationthroughanN-terminalcollagen-likedomain.MBLisakeycomponentinimmuneresp***einthatitcandirectlytriggerneutralizationofinvadingmicroorgani***sbyanAb-independentmechani***.Itbindstosugarsonthesurfaceofbacterial,fungalandparasiticcellsthroughC-terminal,Ca2+-dependentcarbohydrate-recognitiondomains.Mutati***ofhumanMBLareassociatedwithimmunodeficiencyresultingfromareductionintheabilityofthemutantMBLtoinitiatecomplementfixation.Inhuman,twotypesofMBL-associatedserineproteases(MASP-1andMASP-2)andatruncatedformofMASP-2,designated***allMBLassociatedprotein(***AP)orMAp19,complexwithMBLtoactivatethelectinpathwayofthecomplementsystem.ActivatedMASPssubsequentlycle***eandactivatedownstreamcomponentsofthecomplementpathway.TestprincipleThemicrotiterplateprovidedinthiskithasbeenpre-coatedwithanantibodyspecifictoMBP/MBL.Standa***orsamplesarethenaddedtotheappropriatemicrotiterplatewellswithabiotin-conjugatedpolyclonalantibodypreparationspecificforMBP/MBLand***idinconjugatedtoHorseradishPeroxidase(HRP)isaddedtoeachmicroplatewellandincubated.ThenaTMB(3,3'5,5'tetramethyl-benzidine)substratesolutionisaddedtoeachwell.OnlythosewellsthatcontainMBP/MBL,biotin-conjugatedantibodyandenzyme-conjugated***idinwillexhibitachangeincolor.Theenzyme-substratereactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyataw***elengthof450nm&plu***n;2nm.TheconcentrationofMBP/MBLinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.MaterialsandcomponentsReagentQuantityAssayplate1Standard2SampleDiluent1x20mlAssayDiluentA1x10mlAssayDiluentB1x10mlDetectionReagentA1x120ulDetectionReagentB1x120ulWashBuffer1x30ml(25xconcentrate)Substrate1x10mlStopSolution1x10mlPlatesealerfor96wells1x5SamplecollectionandstorageSerum-Useaserumseparatortube(SST)andallowsamplestoclotfor30minutesbeforecentrifugationfor15minutesatapproximately1000xg.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20°Cor-80°C.Pla***a-Collectpla***ausingEDTAorheparinasananticoagulant.Centrifugesamplesfor15minutesat1000xgat2-8°Cwithin30minutesofcollection.Storesamplesat-20°Cor-80°C.***oidrepeatedfreeze-thawcycles.Cellculturesupernatesandotherbiologicalfluids-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20°Cor-80°C.***oidrepeatedfreeze-thawcycles.Samplepreparation-Serum/pla***asamplesrequirea5folddilution.Asuggested5-folddilutionis20uLsample+80uLSampleDiluent.Note:Serum,pla***a,andcellculturesupernatantsamplestobeusedwithin7daysmaybestoredat2-8°C,otherwisesamplesmuststoredat-20°C(≤3months)or-80°C(≤6months)to***oidlossofbioactivityandcontamination.***oidfreeze-thawcycles.Whenperformingtheassayslowlybringsamplestoroomtemperature.Itisrecommendedthatallsamplesbeassayedinduplicate.DONOTUSEHEAT-TREATEDSPECIMENS.Limitati***oftheprocedureFORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.1.Thekitshouldnotbeusedbeyondtheexpirationdateonthekitlabel.2.Donotmixorsubstitutereagentswiththosefromotherlotsorsources.3.Ifsamplesgeneratevalueshigherthanthehigheststandard,furtherdilutethesampleswiththeAssayDiluentandrepeattheassay.Anyvariationinstandarddiluent,operator,pipettingtechnique,washingtechnique,incubationtimeortemperature,andkitagecancausevariationinbinding.4.Thisassayisdesignedtoeliminateinterferencebysolublereceptors,ligands,bindingproteins,andotherfactorspresentinbiologicalsamples.Untilallfactorsh***ebeentestedintheQuantikineImmunoassay,thepossibilityofinterferencecannotbeexcluded.ReagentpreparationBringallreagentstoroomtemperaturebeforeuse.WashBuffer-Ifcrystalsh***eformedintheconcentrate,warmtoroomtemperatureandmixgentlyuntilthecrystalsh***ecompletelydissolved.Dilute30mLofWashBufferConcentrateintodeionizedordistilledwatertoprepare750mLofWashBuffer.Standard-Rec***titutetheStandardwith1.0mLofSampleDiluent.Thisrec***titutionproducesastocksolutionof80ng/mL.Allowthestandardtositforaminimumof15minuteswithgentleagitationpriortomakingserialdiluti***.Theundilutedstandardservesasthehighstandard(80ng/mL).TheSampleDiluentservesasthezerostandard(0ng/mL).DetectionReagentAandB-DilutetotheworkingconcentrationspecifiedontheviallabelusingAssayDiluentAandB(1:100),respectively.AssayprocedureAllowallreagentstoreachroomtemperature.Allthereagentsshouldbemixedthoroughlybygentlyswirlingbeforepipetting.***oidfoaming.Arrangeandlabelrequirednumberofstrips.Prepareallreagents,workingstanda***andsamplesasdirectedintheprevioussecti***.1.Add100uLofStandard,Blank,orSampleperwell.CoverwiththePlatesealer.Incubatefor2hoursat37°C.2.Removetheliquidofeachwell,don’twash.3.Add100uLofDetectionReagentAworkingsolutiontoeachwell.CoverwiththePlatesealer.Incubatefor1hourat37°C.DetectionReagentAworkingsolutionmayappearcloudy.Warmtoroomtemperatureandmixgentlyuntilsolutionappearsuniform.4.Aspirateeachwellandwash,repeatingtheprocessthreetimesforatotalofthreewashes.WashbyfillingeachwellwithWashBuffer(350uL)usingasquirtbottle,multi-channelpipette,manifolddispenserorautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.5.Add100uLofDetectionReagentBworkingsolutiontoeachwell.CoverwithanewPlatesealer.Incubatefor1hoursat37°C.6.Repeattheaspiration/washasinstep4.7.Add90uLofSubstrateSolutiontoeachwell.CoverwithanewPlatesealer.Incubatewithin30minutesat37°C.Protectfromlight.8.Add50uLofStopSolutiontoeachwell.Ifcolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.9.Determinetheopticaldensityofeachwellatonce,usingamicroplatereadersetto450nm.SpecificityThisassayrecognizesrecombinantandnaturalmouseMBP/MBL.Nosignificantcross-reactivityorinterferencewasobserved.SensitivityTheminimumdetectabledoseofmouseMBP/MBListypicallylessthan0.31ng/mL.Thesensitivityofthisassay,orLowerLimitofDetection(LLD)wasdefinedasthelowestproteinconcentrationthatcouldbedifferentiatedfromzero.DetectionRange1.25-80ng/mL.Thestandardcurveconcentrati***usedfortheELISA’swere80ng/mL,40ng/mL,20ng/mL,10ng/mL,5ng/mL,2.5ng/mL,1.25ng/mL.ImportantNote:1.Pleasecarefullyrec***tituteStanda***orworkingDetectionReagentAandBaccordingtotheinstruction,and***oidfoamingandmixgentlyuntilthecrystalsh***ecompletelydissolved.Therec***titutedStanda***canbeusedonlyonce.2.Thewashprocedureiscritical.Insufficientwashingwillresultinpoorprecisionandfalselyelevatedabsorbancereadings.3.Itisrecommendedthatnomorethan32wellsbeusedforeachassayrunifmanualpipettingisusedsincepipettingofallstanda***,specimensandcontrolsshouldbecompletedwithin5minutes.Afullplateof96wellsmaybeusedifautomatedpipettingis***ailable.4.Duplicationofallstanda***andspecimens,althoughnotrequired,isrecommended.5.Whenmixingorrec***titutingproteinsoluti***,always***oidfoaming.6.To***oidcross-contamination,changepipettetipsbetweenadditi***ofeachstandardlevel,betweensampleadditi***,andbetweenreagentadditi***.Also,useseparatereservoirsforeachreagent.7.Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.8.Donotsubstitutereagentsfromonekitlottoanother.Useonlythereagentssuppliedbymanufacturer.Calculationofresults***eragetheduplicatereadingsforeachstandard,control,andsampleandsubtractthe***eragezerostandardopticaldensity.Createastandardcurvebyreducingthedatausingcomputersoftwarecapableofgeneratingafourparameterlogistic(4-PL)curve-fit.Asanalternative,c***tructastandardcurvebyplottingthemeanabsorbanceforeachstandardonthey-axisagainsttheconcentrationonthex-axisanddrawabestfitcurvethroughthepointsonthegraph.ThedatamaybelinearizedbyplottingthelogoftheMBP/MBLconcentrati***versusthelogoftheO.D.andthebestfitlinecanbedeterminedbyregressionanalysis.Thisprocedurewillproduceanadequatebutlessprecisefitofthedata.Ifsamplesh***ebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.Storageoftestkitsandinstrumentation1.Unopenedtestkitsshouldbestoredreferringtothepackagelabelforfrequentuse,andstoredat-20°Cforlongtimestorage.Themicrotiterplateshouldbekeptinasealedbagwithdesiccantstominimizeexposuretodampair.Thetestkitmaybeusedthroughouttheexpirationdateofthekit(sixmonthsfromthedateofmanufacture).Openedtestkitswillremainstableuntiltheexpiringdateshown,provideditisstoredasprescribedabove.2.Openedtestkitswillremainstableuntiltheexpiringdateshown,provideditisstoredasprescribedabove.3.Donotremovemicrotiterplatefromthestoragebaguntilneeded.Unusedstripsshouldbestoredat2-8°Cintheirpouchwiththedesiccantprovided.4.Amicrotiterplatereaderwithabandwidthof10nmorlessandanopticaldensityrangeof0-3ODorgreaterat450nmw***elengthisacceptableforuseinabsorbancemeasurement.5.Usefreshdisposablepipettetipsforeachtransferto***oidcontamination.6.SubstrateSolutioniseasilycontaminated.Ifbluishpriortouse,donotuse.PrecautionTheStopSolutionsuggestedforusewiththiskitisanacidsolution.Weareye,hand,face,andclothingprotectionwhenusingthismaterial.说明书Email:info@索取;13801829628定货；HR-M0373)