人活化凝血因子Ⅻ（FⅫa）英文说明书
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HumanActivatedcoagulationfactorⅫ(FⅫa)FORRESEARCHUSEONLYAssayrange：2.2U/L-80U/L96determinationsPurposeThiskitallowsforthedeterminationofFⅫaconcentrationsinHumanserum,cellculturesupernatesandotherbiologicalfluidsPrincipleoftheassayThekitassayHumanFⅫalevelinthesample,usePurifiedHumanFⅫaantibodytocoatmicrotiterplatewells,makesolid-phaseantibody,thenaddFⅫatowells,CombinedFⅫaantibodywhichWithHRPlabeled,becomeantibody-antigen-enzyme-antibodycomplex,afterwashingCompletely,AddTMBsubstratesolution,TMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.TheconcentrationofHumanFⅫainthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.Materialsprovidedwiththekit1washsolution20ml×1bottle7StopSolution6ml×1bottle2HRP-Conjugatereagent6ml×1bottle8Standard（160U/L）0.5ml×1bottle3Microelisastripplate12well×8strips9Standarddiluent1.5ml×1bottle4Samplediluent6ml×1bottle10Instruction15ChromogenSolutionA6ml×1bottle11Closureplatemembrane26ChromogenSolutionB6ml×1bottle12Sealedbags1Specimenrequirements1.extractassoonaspossibleafterSpecimencollection,andaccordingtotherelevantliterature,and首ldbeexperimentassoonaspossibleaftertheextraction.Ifitcan’t,specimencanbekeptin-20℃topreserve,Avoidrepeatedfreeze-thawcycles.2.Can’tdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.Assayprocedure1.Diluteandaddsample:DiluteOriginaldensityStandardasfollowtable:80U/L5Standard150μlOriginaldensityStandard+150μlStandarddiluent40U/L4Standard150μl5Standard+150μlStandarddiluent20U/L3Standard150μl4Standard+150μlStandarddiluent10U/L2Standard150μl3Standard+150μlStandarddiluent5U/L1Standard150μl2Standard+150μlStandarddiluent2.Addsample:Setblankwellsseparately(blankcomparisonwellsdon’taddsampleandHRP-Conjugatereagent,othereachstepoperationissame).testingsamplewell.addSampledilution40μltotestingsamplewell,thenaddtestingsample10μl(samplefinaldilutionis5-fold),addsampletowells,don’ttouchthewellwallasfaraspossible,andGentlymix.3.Incubate:AfterclosingplatewithClosureplatemembrane,incubatefor30minat37℃.4.Configurateliquid:30-fold(or20-fold)washsolutiondiluted30-fold(or20-fold)withdistilledwaterandreserve.5.Washing:UncoverClosureplatemembrane,discardLiquid,drybyswing,addwashingbuffertoeverywell,stillfor30sthendrain,repeat5times,drybypat.6.Addenzyme:AddHRP-Conjugatereagent50μltoeachwell,exceptblankwell.7.Incubate:Operationwith3.8.Washing:Operationwith5.9.Color:AddChromogenSolutionA50ulandChromogenSolutionB50ultoeachwell,evadethelightpreservationfor10minat37℃10.Stopthereaction:AddStopSolution50μltoeachwell,Stopthereaction(thebluecolorchangetoyellowcolor).11.Assay:takeblankwellaszero,Readabsorbanceat450nmafterAddingStopSolutionandwithin15min.StepsdescriptionStandard,SamplediluentAddStandard,Samplediluent,incubatefor30minat37℃.Wash5time,AddHRP-Conjugatereagent,incubatefor30minat37℃.Wash5times,AddChromogenSolutionAandB,incubatefor10minat37℃.AddStopSolutionReadabsorbanceat450nmwithin15mincalculateCalculateTakethestandarddensityasthehorizontal,theODvalueforthevertical,drawthestandardcurveongraphpaper,FindoutthecorrespondingdensityaccordingtothesampleODvaluebytheSamplecurve,multipliedbythedilutionmultiple,orcalculatethestraightlineregressionequationofthestandardcurvewiththestandarddensityandtheODvalue,withthesampleODvalueintheequation,calculatethesampledensity,multipliedbythedilutionfactor,theresultisthesampleactualdensity.Importantnotes1.Thekittakesoutfromtherefrigerationenvironment首ldbebalanced15-30minutesintheroomtemperature,ELISAplatescoatedifhasnotuseupafteropened,theplate首ldbestoredinSealedbag.2.washingbufferwillCrystallizationseparation,itcanbeheatedthewaterhelpsdissolvewhendilute.Washingdoesnotaffecttheresult.3.addSamplewithsamplerEachstep,Andproofreaditsaccuracyfrequently,avoidstheexperimentalerror.addsamplewithin5min,ifthenumberofsampleismuch,recommendtouseVolley.4.ifthetestingmaterialcontentisexcessivelyhigher(ThesampleODisbiggerthanthefirststandardwell),pleasediluteSample(n-fold),Pleasediluenteandmultipliedbythedilutionfactor.（×n×5）.5.Closureplatemembraneonlylimitsthedisposableuse,toavoidcross-contamination.6.Thesubstrateevadethelightpreservation.7.Pleaseaccordingtouseinstructionstrictly,Thetestresultdeterminationmusttakethemicrotiterplatereaderasastandard.8.Allsamples,washingbufferandeachkindofreject首ldaccordingtoinfectivematerialprocess.9.Donotmixreagentswiththosefromotherlots.Storageandvalidity1．Storage：2-8℃.2．validity：sixmonths
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