Description:
REAADSProteinCAntigenisanenzyme-linkedimmunosorbentassay(ELISA)forthequantitativedeterminationofProteinCAntigenincitratedhumanplasma.
KitComposition:
Reagents
- 12x8anti-humanProteinCantibodycoatedmicrowells.
- 60mlSampleDiluent(blue-greensolution);containssodiumazide.
- 3vialsx0.5mllyophilizedReferencePlasma,withassaysheet
- 12mlanti-humanProteinCHRPConjugate(bluesolution).
- 13mlSubstrate(TMBandH2O2).
- 15mlStoppingSolution(0.36Nsulfuricacid).
- 30mlWashConcentrate(33Xphosphatebufferedsalinewith0.01%Tween20).Note:turbiditymayappearinwashconcentratewhichwillnotaffectcomponentperformanceandshoulddisappearwhenworkingdilutionisprepared.
Storeat2–8°C.DoNotFreeze.
Materialsrequiredbutnotsupplied
- ProteinCControlPlasma.ReconstituteControlPlasmaselectedforusefollowingmanufacturer’sinstructions,andstoreasrecommended.
- Reagentgradewater(1L)topreparePBS/Tween20washsolution,toreconstituteReferencePlasma,andtozeroorblanktheplatereaderduringthefinalassaystep.
- Graduatedcylinders
- Precisionpipettorscapableofdeliveringbetween5and1000microliters,withappropriatetips
- Miscellaneousglasswareappropriateforsmallvolumehandling
- Flaskorbottle,1liter
- Washbottles,preferablywiththetippartiallycutbacktoprovideawidestream,oranautomatedorsemi-automatedwashingsystem
- Disposablegloves,powder-freerecommended
- PlatereADIngspectrophotometercapableofreadingabsorbanceat450nm(witha650nmreferenceifavailable)
- Multichannelpipettorscapableofdeliveringto8wellssimultaneously
- Microdilutiontubesforpatientsamplepreparation
MeasurementPrinciple:
Principle
TheProteinCAntigenassayisasandwichELISA.AcaptureantibodyspecificforhumanProteinCiscoatedto96-microwellpolystyreneplates.Dilutedpatientplasmaisincubatedinthewells,allowinganyavailableProteinCtobindtotheanti-humanProteinCantibodyonthemicrowellsurface.Theplatesarewashedtoremoveunboundproteinsandotherplasmamolecules.BoundProteinCisquantitatedusinghorseradishperoxidase(HRP)conjugatedanti-humanProteinCdetectionantibody.Followingincubation,unboundconjugateisremovedbywashing.Achromogenicsubstrateoftetramethylbenzidine(TMB)andhydrogenperoxide(H2O2)isaddedtodevelopacoloredreaction.Theintensityofthecolorismeasuredinopticaldensity(O.D.)unitswithaspectrophotometerat450nm.ProteinCAntigenrelativepercentconcentrationsinpatientplasmaaredeterminedagainstacurvepreparedfromthereferenceplasmaprovidedwiththekit.
Procedure
DilutedcitratedpatientplasmaandcontrolsareincubatedinmicrowellscoatedwithcaptureantibodyspecificforhumanProteinC.Duringanincubationperiod,patientProteinCisallowedtobindtothesurface.
Followingawashtoremoveanyunboundplasma,horseradishperoxidase(HRP)conjugatedanti-humanProteinCdetectionantibodyisaddedtothewells.Afterwashingtoremoveunboundconjugate,achromogenicsubstrateisadded,resultinginasolublecoloredproductthatismeasuredinaspectrophotometerat450nmfollowingtheadditionofastopsolution.
PatientProteinClevelsaredeterminedfromasixpointcurvepreparedfromthereferenceplasmaprovidedinthekit.Totalincubationtimeis60minutes.
AssayProcedure:
1.Removeanymicrowellstripsthatwillnotbeusedfromtheframeandstoretheminthebag provided.
2.Assayeachreferenceplasmadilutioninduplicate.Duplicatedeterminationsarealsorecommended forpatientandcontrolsamples.Onewellshouldberunasareagentblank;samplediluentwithout serumisaddedtothewellasexplainedinstep7ofthissection.Thiswellistreatedthesameasa controlorpatientsampleinsubsequentassaysteps.Awaterblankwellshouldbeincludedwith eachplate;itistoremainemptyuntil200µlofreagentgradewaterisaddedatthecompletionof theassay,immediatelypriortoreadingtheplate.Thewaterblankwellistobeusedtozerotheplate reader.
3.Pre-diluteallplasmas(1:2dilutioninSampleDiluent)asfollows: Referenceplasma:add100µlreferenceplasmato100µlSampleDiluent Controlandpatientsamples:add20µlplasmato20µlSampleDiluent Mixwell.Thesepre-dilutionsareutilizedinpreparingtheworkingdilutionsinsteps4and5.
4.Usingthe1:2referenceplasmadilutionfromstep3,preparesixworkingreferencedilutionsas describedbelow.
| VolumeReference Plasma(1:2) | VolumeSample Diluent | *ReferenceLevel | ||
| 30μl | + | 500μl | = | 150 |
| 20μl | + | 500μl | = | 100 |
| 15μl | + | 500μl | = | 75 |
| 10μl | + | 500μl | = | 50 |
| 10μl | + | 1000μl | = | 25 |
| 10μl | + | 2000μl | = | 12.5 |
| *Referencelevelvaluetobeusedforconstructingreferencecurveonly | ||||
5.Prepareworkingdilutionsofcontrolandpatientsamplesbyadding20µlofpredilutedplasma(1:2 dilutionfromstep3)to500µlSampleDiluent.(Note:thesedilutionscorrespondtothe100% referenceplasmadilution.)
6.Mixthoroughly,andadd100µloftheworkingdilutions(referenceplasmas,controlsandpatient samples)totheappropriatemicrowells.
7.Add100µlofSampleDiluenttothereagentblankwell.Leavethewaterblankwellempty.
8.Incubate40minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthe microwellsanddumpthesamplefluid.Donotallowsamplestocontaminateothermicrowells.
9.Wash4timeswithworkingwashsolution(PBS/Tween20).Eachwellshouldbefilledwithwash solutionperwash.Washsolutionintheemptywellintendedtoserveasawaterblankwillnot interferewiththeprocedure.Invertmicrowellsbetweeneachwashtoemptyfluid.Useasnapping motionofthewristtoshaketheliquidfromthewells.Theframemustbesqueezedatthecenteron thetopandbottomtoretainmicrowellmodulesduringwashing.Blotonabsorbentpapertoremove residualwashfluid.Donotallowwellstodryoutbetweensteps.
10.Add100µlConjugate(blue)toeachwell(exceptthewaterblankwell).
11.Incubatefor10minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthe microwellsanddumptheconjugatesolution.
12.Wash4timeswithworkingwashsolution(PBS/Tween20)asinstep9.Washsolutioninthewater blankwelldoesnotinterferewiththeprocedure.Useasnappingmotiontodraintheliquid,andblot onabsorbentpaperafterthefinalwash.Donotallowthewellstodryout.
13.Add100µlSubstratetoeachwell(exceptforthewaterblankwell)andincubatefor10minutesat roomtemperature.Addthesubstratetothewellsatasteadyrate.Bluecolorwilldevelopinwells withpositivesamples.
14.Add100µlStoppingSolution(0.36Nsulfuricacid)toeachwell(exceptforthewaterblankwell)to stoptheenzymereaction.BesuretoaddStoppingSolutiontothewellsinthesameorderandatthe samerateastheSubstrateSolutionwasadded.BlueSubstratewillturnyellowandcolorlesssubstrate willremaincolorless.DonotaddStoppingSolutiontothewaterblankwell.Instead,add200µlof reagentgradewatertothewaterblankwell.Blankorzerotheplatereaderagainstthewaterblank well.ReadtheO.D.ofeachwellat 450nm,againsta650nmreferencefilter(ifavailable).Forbestresults,theO.D.valuesshouldbemeasuredwithin30minutesaftertheadditionofStoppingSolution.
Performance:
ClinicalPerformance
PlasmasamplesfromhealthyblooddonorsandfrompatientswithahistoryofthrombosisweretestedtodefineandcomparetheclinicalperformanceofREAADSProteinCELISAwithawellestablished,commerciallyavailableProteinCAntigenRocketEIDmethod.Asshowninthetable,theresultscorrelatedwell,andwereshowntobestatisticallysimilarbysinglefactorAnova.
TechnicalPerformance
Intra-assayprecisionofREAADSProteinCELISAis7.0%whileintra-assayprecisionis7.5%Linearity,expressedasthecoefficientofdetermination(r2)is0.992withameanaccuracyof99.4%REAADSProteinCELISAisarapid,convenient,highlyaccurateandprecisemethodforthequantitativedeterminationofProteinClevelsinhumanplasma.
Background:
ProteinCisavitaminK-dependentproteinsynthesizedprimarilybyhepatocytesintheliverandplaysanimportantphysiologicroleintheProteinCAnticoagulantSystem.ProteinC,thrombinfrombloodclots,andendothelialcells,throughcomplexinteractionswithotherfactorsofthecoagulationcascade,contributetothemaintenanceofnormalhemostaticmechanismsbydown-regulatingclotformationandbypromotingfibrinolysis.TheProteinCAnticoagulantSystemisactivatedbythebindingofthrombintothrombomodulin,atransmembraneproteinreceptoronendothelialcells.Thethrombin-thrombomodulinbindingonendothelialcellmembranesactivatescirculatingProteinC.ActivatedProteinCbindstoProteinSonthemembraneofendothelialcellsorplatelets.InthisProteinC-ProteinScomplex,activatedProteinCisnowcapableofinactivatingcoagulationfactorsVaandVIIIa,down-regulatingclotformation.ActivatedProteinCalsoenhancesthefunctionoftissueplasminogenactivator(TPA)bydissociatingthismoleculefromitsinhibitor,plasminogenactivatorinhibitor-1(PAI-1),therebyfacilitatingclotdissolutionorfibrinolysis.
ProteinCdeficiency,eithercongenitaloracquired,mayleadtoseriousthromboticeventssuchasthrombophlebitis,deepveinthrombosis,orpulmonaryembolism.Patientswithacongenitalheterozygousdeficiencymaypresentwithvenousthrombosisinyoungadulthood,whilepatientswiththerarehomozygousdeficiencypresentwithmassivethrombosis(purpurafulminans)duringtheneonatalperiod.TheprevalenceofProteinCdeficiencyinthegeneralpopulationhasbeenestimatedat1in300.Inyoungerpatients(<40-45years)withrecurrentvenousthrombosis,thefrequencyofProteinCdeficienciesmaybeashighas10to15%.AcquiredProteinCdeficiencymaybeseeninliverdisease,extensivethromboticepisodes,surgery,oralanticoagulanttherapy,antiphospholipidsyndrome,etc.AdecreasedProteinCactivityinplasmamaybetheresultoflowconcentrationsandfunction(typeI)oronlylowfunction(typeII).
ThelaboratorydiagnosisofProteinCdeficiencymayrequirebothquantitativeandqualitative(functional)determinations.QuantitativedeterminationsofProteinCAntigenarebasedonimmunologicproceduressuchasradialimmunodiffusioningel,Laurellrocketimmunoelectrophoresisandenzyme-linkedimmunosorbentassay(ELISA).ELISAproceduresarelesslaborintensiveandofferseveraladvantagesincludingmoreobjective,accurateandreproducIBLeresults.Inaddition,ELISAallowsautomationwithcommonlyavailablelaboratoryinstruments.
