Description:

TheREAADSvWFActivitytestkitisanELISAforthequantitativedetectionofvonWillebrandFactoractivityincitratedhumanplasma.Theassayutilizesapurifiedmurineanti-vWFmonoclonalantibodywhichrecognizesafunctionalepitopeonthevWFmoleculetoassessvWFactivitylevels.

Resultsarereportedinpercent(%)ofnormal,relativetoacalibratorthathasbeenstandardizedagainstthethirdInternationalStandardforFactorVIIIandvonWillebrandFactorinPlasma(91/666).

Features:

• UtilizesamonoclonalantibodywhichrecognizesafunctionalepitopeonvWFmolecule
• Totalincubationtimeis40minutes
• ConvenientELISAProcedure
• Easytouse

KitComposition:

Reagents

• 12x8stABIlizedantibodycoatedmicrowells(12stripsof8breakawaywells),withframe.WellsarecoatedwithamonoclonalantibodyspecificforthefunctionalregionofvWF.(mouse)
• 60mlSampleDiluent(blue-greensolution);*
• 3x0.5mllyophilizedReferencePlasmaforpreparationofreferencecurve,withassaysheet.
• 12mlHRP-Conjugatedanti-humanvWFAntibodySolution(bluesolution).(leporine
• 13mlSubstrate(TMB/H₂O₂).
• 15mlStoppingSolution(0.36Nsulfuricacid).
• 30mlWashConcentrate[33Xphosphatebufferedsaline(PBS)with0.01%Tween20].Note:turbiditymayappearinwashconcentratewhichwillnotaffectcomponentperformanceandshoulddisappearwhenworkingdilutionisprepared.

Storeat2-8°C.DoNotFreeze.

Materialsrequiredbutnotsupplied

VWF:ActControlPlasma.ReconstituteControlPlasmaselectedforusefollowingmanufacturer’sinstructions,andstoreasrecommended.

• Reagentgradewater(1L)topreparePBS/Tween20washsolution,toreconstituteReferencePlasma,andtozeroorblanktheplatereaderduringthefinalassaystep.
• Graduatedcylinders
• Precisionpipettorscapableofdeliveringbetween5and1000µl,withappropriatetips
• Miscellaneousglasswareappropriateforsmallvolumehandling
• Flaskorbottle,1liter
• Washbottles,preferablywiththetippartiallycutbacktoprovideawidestream,oranautomatedorsemi-automatedwashingsystem
• Disposablegloves,powder-freerecommended
• PlatereADIngspectrophotometercapableofreadingabsorbanceat450nm(witha650nmreference,ifavailable)
• Multichannelpipettorscapableofdeliveringto8wellssimultaneously
• Microdilutiontubesforpatientsamplepreparation

MeasurementPrinciple:

Principle

TheREAADSvonWillebrandFactorActivityTestisanELISAforthequantitativedetectionofvWFactivityincitratedhumanplasma.Theassayutilizesapurifiedmurineanti-vWFmonoclonalantibodywhichrecognizesafunctionalepitopeonthevWFmoleculetoassessvWFactivitylevels.

Resultsarereportedinpercent(%)ofnormal,relativetoacalibratorthathasbeenstandardizedagainstthethirdInternationalStandardforFactorVIIIandvonWillebrandFactorinPlasma(91/666).

Procedure

DilutedcitratedpatientplasmaandcontrolsareincubatedinmicrowellscoatedwithmonoclonalcaptureantibodyallowingpatientvWFtobindtothesurface.Followinganincubationperiod,thewellsarewashed,andahorseradishperoxidase(HRP)conjugatedanti-humanvWFdetectionantibodyisadded.Afterincubation,thewellsarewashed,substrateisadded,andcolordevelopmentismeasuredinaspectrophotometerat450nmfollowingtheadditionofastopsolution.PatientvWF:Actlevelsaredeterminedfromasix-pointcurvepreparedfromthereferenceplasmaprovidedinthekit.Totalincubationtimeis40minutesatroomtemperature.

AssayProcedure:

1.Removeanymicrowellstripsthatwillnotbeusedfromtheframeandstoretheminthebagprovided.

2.Assayeachreferenceplasmadilutioninduplicate.Duplicatedeterminationsarealsorecommendedfor patientandcontrolsamples.Onewellshouldberunasareagentblank;samplediluentwithoutplasma isaddedtothewellasexplainedinstep6ofthissection.Thiswellistreatedthesameasapatient sampleinsubsequentassaysteps.Awaterblankwellshouldbeincludedwitheachplate;itisto remainemptyuntil200µlofreagentgradewaterisaddedatthecompletionoftheassay,immediately priortoreadingtheplate.Thewaterblankwellistobeusedtozerotheplatereader.

3.UsingtheReferencePlasmaprovidedwiththekit,preparefivereferenceplasmadilutionsasdescribed below.

4.Preparea1:21dilutionofeachpatientsampleandcontrolplasmaselectedforuseinSampleDiluent (blue-greensolution);e.g.25µlsampleaddedto500µlSampleDiluent.Mixthoroughly.

5.Add100µlofthedilutions(referenceplasmasx5,patientsamplesandcontrols)totheappropriate microwells.

6.Add100µlofSampleDiluenttothereagentblankwell.Leavethewaterblankwellempty.

7.Incubate15minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthe microwellsandemptythefluid.Donotallowsamplestocontaminateothermicrowells.

8.Wash4timeswithworkingwashsolution(PBS/Tween20).Eachwellshouldbefilledwithwash solutionperwash.Washsolutionintheemptywellintendedtoserveasawaterblankwillnotinterfere withtheprocedure.Invertmicrowellsbetweeneachwashtoemptyfluid.Useasnappingmotionofthe wristtoshaketheliquidfromthewells.Theframemustbesqueezedatthecenteronthetopand bottomtoretainmicrowellmodulesduringwashing.Blotonabsorbentpapertoremoveresidualwash fluid.Donotallowwellstodryoutbetweensteps.

9.Add100µlHRP-ConjugatedAntibodySolution(blue)toeachwell(exceptthewaterblankwell).

10.Incubatefor15minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthe microwellsandemptytheconjugatesolution.

11.Wash4timeswithworkingwashsolution(PBS/Tween20)asinstep8.Washsolutioninthewater blankwellwillnotinterferewiththeprocedure.Useasnappingmotiontodraintheliquid,andbloton absorbentpaperafterthefinalwash.Donotallowthewellstodryout.

12.Add100µlSubstratetoeachwell(exceptforthewaterblankwell)andincubatefor10minutesat roomtemperature.Addthesubstratetothewellsatasteadyrate.Bluecolorwilldevelopinwellswith positivesamples.

13.Add100µlStoppingSolution(0.36Nsulfuricacid)toeachwell(exceptforthewaterblankwell)tostop theenzymereaction.BesuretoaddStoppingSolutiontothewellsinthesameorderandatthesame rateastheSubstratewasadded.Bluesubstratewillturnyellowandcolorlesssubstratewillremain colorless.DonotaddStoppingSolutiontothewaterblankwell.Instead,add200µlreagentgrade watertothewaterblankwell.Blankorzerotheplatereaderagainstthewaterblankwell.Readthe O.D.ofeachwellat450nm,againsta650referencefilter(ifavailable).Forbestresults,theO.D.valuesshouldbemeasuredwithin30minutesaftertheadditionofStoppingSolution.

Performance:

ClinicalPerformance

100citratedplasmasamplesfromhealthyindividualswereassayedforvonWillebrandActivityusingtheREAADSkit.90%oftheresultsforthispopulationwere≥50%activity.ComparableresultsareseenbetweentheREAADSvonWillebrandFactorActivityTestKit,andanothercommerciallyavailablevWFactivityELISAkitwhentestingtype2vWDsamples.UsingtheREAADSvonWillebrandFactorActivityTestKit,themeanvonWillebrandFactorActivityforthepopulationis26.5%withresultsrangingfrom1.7-59.8%.Usinganothercommerciallyavailablekit,thepopulationmeanwas19.7%,witharangeof1.5-147.3%.Whentheresultswerecompared,thevaluesreportedbythetwomethodsareshowntobestatisticallysimilar.

TechnicalPerformance

Precisionwithinalotwasdeterminedbytesting14commerciallypreparedplasmasamples,induplicatedilutions,acrossthreeplatelots.ThemeanCVforduplicatesoverthreeplatelotswas5.2%.Theprecisionbetweenlotswasdeterminedbycomparingthevaluesrecoveredforeightdifferentcontrolsamplesontwolots.Eachoftheeightsampleswastestedinduplicateineachassay.Themeaninter-assayCVwas8.3%.Thelowerlimitofdetection,ascalculatedfromthereagentblankplus3standarddeviations,using3kitlots,was3.1%.

Background:

vonWillebrandFactor(vWF),aprocoagulantprotein,playstwoimportantrolesinhemostasis:

1. Mediatesplateletadhesiontositesofvascularinjury.
2. ProtectsfactorVIIIfromproteolyticcleavageincirculation.InvonWillebrandDisease(vWD),whichiscausedbyeitherquantitative(TypeI)orqualitative(TypeII)deficienciesinvWF,abnormalbleedingmayresultduetoimpairedplateletfunctionandclottingfactorinhibition.vWDisthemostcommoninheritedbleedingdisorder,andisclinicallycharacterizedbyeasybruisingorprolongedbleedingfrommucosalsurfaces.Whileapproximately80%ofvWDpatientshaveTypeIdeficiency,bothquantitative(antigenic)andqualitative(functional)assaysmayberequiredforalaboratorydiagnosisofvWD.

vonWillebrandFactorAntigen(vWF:AgorFactorVIII-relatedprotein)isaplasmaproteinfoundincirculationcombinedbynon-covalentinteractionswithFactorVIII(FVIII:C),apro-coagulantproteinalsoknownastheanti-hemophilicfactor.Thesetwoproteinsshowdistinctbiochemicalandfunctionalpropertiesaswellasdifferentantigenicdeterminants;theirplasmalevelsmayvaryindependentlyofeachother.DeficiencyofFVIIIcausesclassichemophiliawhiledeficiencyofvWFcausesvonWillebranddisease.MostofVWF:Agissynthesizedandstoredbyendothelialcellswhile15-20%issynthesizedbymegakaryocytesandstoredincirculatingplatelets.AvWF:Agunithasamolecularweightofabout250kDandtendstopolymerizeincirculation,withmultimersranginginsizefrom850kDtoaslargeas15×106D.

vWFplaysaveryimportantroleinhemostasis;itprotectsFVIIIfromproteolyticcleavageincirculationandhelpsplateletstoaggregateortoadheretositesofvasculardamage.Theinvivohalf-lifeofFVIII:CwithoutvWFisshortenedfrom10-12hourstoafewminutes.Thesetwomechanismspreventbleeding.vonWillebranddiseaseischaracterizedbyadeficiencyordefectofVWF.DecreasedvWFactivityinplasmacanbetheresultoflowconcentrations(quantitativeortypeIdefect)orfunctionalchangesofvWF(qualitativeortypeIIdefect).vonWillebranddiseaseisthemostcommoninheritedbleedingdisorderandischaracterizedbyeasybruisingandprolongedbleedingfrommucosalsurfaces.TheprevalenceofVonWillebranddiseasehasbeenestimatedtobe1-3%ofthegeneralpopulation.Greaterthan70%ofVonWillebranddiseasepatientshaveatypeIdeficiencywhileapproximately20%haveatypeIIdeficiency.

ThelaboratorydiagnosisofvonWillebranddiseasemayrequirebothquantitativeandqualitative(functional)determinationstodifferentiatethetwopredominantsubtypesofthedisease,typeIandtypeII.TheclassificationofVonWillebranddiseaseintosubtypesisimportantindeterminingthecourseofclinicaltreatment.