Description:

REAADSMonoclonalFreeProteinS usesamonoclonalantibodyspecificforfreeProteinStomeasurementfreeProteinSlevelsincitratedhumanplasma.Nopretreatmentofsampleswithpolyethyleneglycol(PEG)isrequired.Resultsarereportedinpercent(%)ofnormal,relativetoareferenceplasmathathasbeenstandardizedagainsttheSecondaryStandardforCoagulation/InternationalSocietyonThrombosisandHemostasis (SSC/ISTH)preparation,whichiscalibratedtoWorldHealthOrganization(WHO)standards.

Advantages:

• UtilizesamonoclonalantibodyspecificforFreeProteinS
• ConvenientELISAprocedure
• Objective,accurateandreproducIBLe
• Reagentcompletekit
• Totalincubatetime:60minutesatroomtemperature

KitComposition:

Reagents

• 12x8MouseMonoclonalantibodytohumanFreeProteinScoatedmicrowells.
• 60mlSampleDiluent(blue-greensolution);containssodiumazide.
• 3x0.5mlLyophilizedReferencePlasma,withassaysheet.
• 12mlRabbitAnti-humanProteinSHRPConjugate(redsolution).
• 13mlSubstrate(TMBandH₂O₂).
• 15mlStoppingSolution(0.36Nsulfuricacid).
• 30mlWashConcentrate(33XPBSwith0.01%Tween20).Note:turbiditymayappearinwashconcentratewhichwillnotaffectcomponentperformanceandshoulddisappearwhenworkingdilutionisprepared.

Storeat2–8°C.DoNotFreeze.

MaterialsRequiredbutnotSupplied

• FreeProteinSControlPlasma.ReconstituteControlPlasmaselectedforusefollowingmanufacturer’sinstructions,andstoreasrecommended.
• Reagentgradewater(1L)topreparePBS/Tweenwashsolution,toreconstituteReferencePlasma,andtozeroorblanktheplatereaderduringthefinalassaystep.
• Graduatedcylinders
• Precisionpipettorscapableofdeliveringbetween5and1000microliters,withappropriatetips
• Miscellaneousglasswareappropriateforsmallvolumehandling
• Flaskorbottle,1liter
• Washbottles,preferablywiththetippartiallycutbacktoprovideawidestream,oranautomatedorsemi-automatedwashingsystem
• Disposablegloves,powder-freerecommended
• PlatereADIngspectrophotometercapableofreadingabsorbanceat450nm(witha650nmreference,ifavailable)
• Multichannelpipettorscapableofdeliveringto8wellssimultaneously
• Microdilutiontubesforpatientsamplepreparation
• Centrifuge

PrincipleandProcedure:

Principle

REAADSMonoclonalFreeProteinSELISAusesamonoclonalantibodyspecificforfreeProteinStomeasurementfreeProteinSlevelsincitratedhumanplasma.Nopretreatmentofsampleswithpolyethyleneglycol(PEG)isrequired.Resultsarereportedinpercent(%)ofnormal,relativetoareferenceplasmathathasbeenstandardizedagainsttheSecondaryStandardforCoagulation/InternationalSocietyonThrombosisandHemostasis (SSC/ISTH)preparation,whichiscalibratedtoWorldHealthOrganization(WHO)standards.

Procedure

DilutedcitratedpatientplasmaisincubatedinmicrowellscoatedwithamonoclonalcaptureantibodyspecificforfreeProteinS,allowingpatientfreeProteinStobindtothesurface.Afterwashingtoremoveunboundplasmaproteins,HRP-conjugatedpolyclonalanti-humanProteinSdetectionantibodyisadded,whichattachestothesurfaceboundfreeProteinSantigenduringasecondincubation.Thewellsarewashed,andachromogenicsubstrateisadded,resultinginasolublecoloredproductthatismeasuredinaspectrophotometerat450nmaftertheadditionofstopsolution.TheconcentrationoffreeProteinSinthetestsampleisdeterminedfromastandardcurvepreparedfromthereferenceplasmaprovidedinthekit.Totalassayincubationtimeis60minutesatroomtemperature.

Performance:

ClinicalPerformance

Theclinicalperformancewasdeterminedbytestingplasmasamplesfrom35healthyindividualsand20patientswithknownProteinSdeficiencywithREAADSMonoclonalFreeProteinSassayandREAADSProteinSAntigentestkit(PEGmethod).Asshowninthetable,agoodcorrelationwasseenbetweenthetwomethodsforthecombinedtestpopulation(r=0.980),withaPvalueof0.739bysinglefactorAnova.

TechnicalPerformance

Intra-assayprecision,expressedin%CV,was4.7%whensamplesranginginvaluefrom6–150%weretestedinduplicatewithREAADSMonoclonalFreeProteinSassay.Inter-assayprecisionwasshowntobe5.2%.AccuracywasdemonstratedbytestingtherecoveryofplasmasamplesspikedwithknownlevelsoffreeProteinS;themean%recoverywas101.2%acrossthreeproductionlots.Linearitywasdeterminedbylinearregressionofthelog-logcurveexpressedasthecoefficientofdetermination(r²)=0.994.

REAADSMonoclonalFreeProteinSAssayisarapid,accurateandprecisemethodforthedeterminationoffreeProteinSlevelsinhumanplasma,offeringimprovedspecificity,convenienceandsignificanttimesavingsovertraditionalmethodthatrequirePEGprecipitation.

Background:

ProteinSisavitaminK-dependentproteinsynthesizedintheliver,vascularendothelium,andmegakaryocytes,whichplaysanimportantphysiologicroleintheProteinCAnticoagulantSystem.Thisanticoagulantsystemisoneofthemajorregulatorsofhemostasisbyinhibitingclotformationandbypromotingfibrinolysis.ProteinSfunctionsasacofactorforactivatedProteinConthevascularmembranetofacilitatethedegradationofclottingfactorsVaandVIIIa,down-regulatingclotformation.Innormalplasmaapproximately40%ofProteinScirculatesasafreemolecule,while60%iscomplexedwithC4b,aplasmaproteinoftheclassicalcomplementpathway.OnlyFreeProteinSisfunctionallyactiveandabletobindtoactivatedProteinC,whilethecomplexedformofProteinSisnot.

ProteinSdeficiency,eithercongenitaloracquired,mayleadtoseriousthromboticeventssuchasthrombophlebitis,deepveinthrombosis,orpulmonaryembolism.TheprevalenceofProteinSdeficiencyhasbeenestimatedtobelessthan1caseper300inthegeneralpopulation.Two-thirdsofpatientswithacongenitaldeficiencyofProteinS(levelslessthan50%ofnormal)maypresentwithvenousthrombosisinyoungadulthood.Inyoungpatients(<35years)withahistoryofthrombosis,theprevalencemaybeashighas15to18%.AcquiredProteinSdeficiencymaybeseenduringpregnancy,oralcontraceptiveororalanticoagulanttherapy,liverdisease,diabetesmellitus,postoperativecomplications,septicemia,andvariousinflammatorysyndromes.AdecreasedProteinSactivityinplasmamaybetheresultoflowconcentrationsorabnormalfunctionoftheProteinSmolecule.

ThelaboratorydiagnosisofProteinSdeficiencymayrequirebothquantitativeandqualitative(functional)determinations.QuantitativedeterminationsofProteinSAntigenarebasedonimmunologicproceduressuchasradialimmunodiffusioningel,Laurellrocketimmunoelectrophoresis,andenzyme-linkedimmunosorbentassay(ELISA).ELISAproceduresarelesslaborintensiveandofferseveraladvantagesincludingmoreobjective,accurate,andreproducibleresults.Inaddition,theELISAformatallowsautomationwithcommonlyavailablelaboratoryinstrumentation.

MeasurementofplasmalevelsofbothTotalandFreeProteinSareusefulindeterminingthetypeofdefectinpatientswithProteinSdeficiency.Historically,ELISAproceduresmeasuringProteinSusedapolyclonalantibodyspecifictoboththefreeandboundformsofProteinS.Theadditionofpolyethyleneglycol(PEG)toprecipitatetheboundProteinSinthepatientsamplealloweddeterminationoflevelsoffreeProteinS.WhilethePEGprecipitationprocedureallowsthemeasurementofFreeProteinS,itisnon-specific,timeconsuming,anddifficulttoperformaccurately.ThisassayutilizesamonoclonalantibodyspecificforFreeProteinSinanELISAformattomeasureFreeProteinSdirectly,withoutPEGprecipitation.