Description:
REAADSProteinSAntigenisanenzyme-linkedimmunosorbentassay(ELISA)forthequantitativedeterminationofTotalandFreeProteinSAntigenincitratedhumanplasma.
Advantages:
- TotalandFreeproteinS
- ConvenientELISAprocedure
- Reagentcompletekit– noextraexpenseforadditionalreagents–costeffective
- Sixpointreferencecurve
- Accuratemeasurementofaspecificprotein
- Noreconstitutionrequired,reducingriskofdilutionerrors
- Kitreagentsoptimizedformoreaccurateresults
- Plateandcomponentsarestableafteropeningthroughshelf-life–costeffective
- CalibratedforProteinC,SandvWFassaystoISTHstandardsandoptimizedforeachlotformoreaccurateresults
- Assayformatsavailableforbothmanualandselectautomatedplatforms
- Totalincubationtime:60minutes
KitComposition:
Reagents
- 12x8anti-humanProteinSantibodycoatedmicrowells
- 60mlSampleDiluent(blue-greensolution);containssodiumazide.
- 3vialsx0.5mllyophilizedReferencePlasma,withassaysheet.
- 12mlanti-humanProteinSHRPConjugate(redsolution).
- 13mlSubstrate(TMBandH2O2).
- 15mlStoppingSolution(0.36Nsulfuricacid).
- 30mlWashConcentrate(33XPBSwith0.01%Tween20).Note:turbiditymayappearinwashconcentratewhichwillnotaffectcomponentperformanceandshoulddisappearwhenworkingdilutionisprepared.
- 2mlFreeProteinSReagent(PEG).
Storeat2–8°C.DoNotFreeze.
MaterialsRequiredbutnotSupplied
- ProteinSControlPlasma(Totaland/orFree).ReconstituteControlPlasmaselectedforusefollowingmanufacturer’sinstructions,andstoreasrecommended.
- Reagentgradewater(1L)topreparePBS/Tweenwashsolution,toreconstituteReferencePlasma,andtozeroorblanktheplatereaderduringthefinalassaystep.
- Graduatedcylinders
- Precisionpipettorscapableofdeliveringbetween5and1000microliters,withappropriatetips
- Miscellaneousglasswareappropriateforsmallvolumehandling
- Flaskorbottle,1liter
- Washbottles,preferablywiththetippartiallycutbacktoprovideawidestream,oranautomatedorsemi-automatedwashingsystem
- Disposablegloves,powder-freerecommended
- PlatereADIngspectrophotometercapableofreadingabsorbanceat450nm(witha650nmreferenceifavailable)
- Multichannelpipettorscapableofdeliveringto8wellssimultaneously
- Microdilutiontubesforpatientsamplepreparation
- Centrifuge
PrincipleandProcedure:
Principle
TheREAADSProteinSAntigentestkitisadoubleantibodycaptureassayformeasuringtotalandfreeProteinSlevelsinhumanplasma,expressedinrelativepercent(%)ofnormal.TheassayisintendedtobeusedasanaidinthediagnosisofProteinSdeficiencyinpatientswiththromboticdisorders.TheREAADSProteinSAntigentestkitwillaccuratelydetectantigenlevelsaslowas5%ofnormal.
Procedure
DilutedcitratedpatientplasmaisincubatedinmicrowellscoatedwithcaptureantibodyspecificforhumanProteinS,allowingpatientProteinStobindtothesurface.ThehumanProteinSdetectionantibodyisadded.Afterincubation,thewellsarewashed,substrateisaddedandcolordevelopmentismeasuredinaspectrophotometerat450nmfollowingtheadditionofastopsolution.PatientProteinSlevelsaredeterminedfromasix-pointcurvepreparedfromthereferenceplasmaprovidedinthekit.FreeProteinSlevelscanbemeasuredsimultaneouslybytestingsamplesthathavebeenpretreatedwithPEG,followingthesameassayprocedure.Totalincubationtimeis60minutes.
Performance:
ClinicalPerformance
PlasmasamplesfromhealthyblooddonorsandfrompatientswithahistoryofthrombosisweretestedtodefineandcomparetheclinicalperformanceofREAADSProteinSELISAwithawellestablished,commerciallyavailableProteinSAntigenRocketEIDmethod.Asshowninthetable,theresultscorrelatedwell,andwereshowntobestatisticallysimilarbysinglefactorANOVA.
| REAADS | RocketEID | |||
| TotalProteinS | Healthy | Mean | 105% | 98% |
| Range | 64–150% | 65–143% | ||
| Patients | Mean | 59% | 58% | |
| Range | 23–140% | 23–148% | ||
| Free ProteinS | Healthy | Mean | 97% | 102% |
| Range | 61–162% | 62–160% | ||
| Patients | Mean | 47% | 52% | |
| Range | 12–115% | 20–127% | ||
| Correlation(r)=0.934;Pvalue=0.346 | ||||
TechnicalPerformance
Intra-assayprecisionis10.1%forREAADSTotalProteinS,and6.6%fortheFreeProteinSassay.Inter-assayprecisionis11.0%forTotal,and10.5%forFreeProteinS.Linearity,expressedasthecoefficientofdetermination(r2)is0.985forTotal,and0.992forFreeProteinS.Meanaccuracyis101%forTotal,and98%forFreeProteinSassays.REAADSProteinSELISAisarapid,convenient,highlyaccurateandprecisemethodforthequantitativedeterminationofProteinSlevelsinhumanplasma.
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%.7AcquiredProteinSdeficiencymaybeseenduringpregnancy,oralcontraceptiveororalanticoagulanttherapy,liverdisease,diabetesmellitus,postoperativecomplications,septicemiaandvariousinflammatorysyndromes.AdecreasedProteinSactivityinplasmamaybetheresultoflowconcentrationsorabnormalfunctionoftheProteinSmolecule.
ThelaboratorydiagnosisofProteinSdeficiencymayrequirebothquantitativeandqualitative(functional)determinations.QuantitativedeterminationsofProteinSAntigenarebasedonimmunologicproceduressuchasradialimmunodiffusioningel,Laurellrocketimmunoelectrophoresisandenzyme-linkedimmunosorbentassay(ELISA).9,10ELISAproceduresarelesslaborintensiveandofferseveraladvantagesincludingmoreobjective,accurateandreproducIBLeresults.Inaddition,ELISAallowsautomationwithcommonlyavailablelaboratoryinstrumentation.MeasurementofplasmalevelsofbothTotalandFreeProteinSareusefultodeterminethetypeofdefectinpatientswithProteinSdeficiency.
