Description:

TheTechnozym®tPACombiActibind®ELISAtestkitmeasurest-PAfunctionalactivity,non-complexedt-PAantigenandt-PA-PAI-1complexes.Itisnotaffectedbythepresenceofotherplasminogenactivators.Themeasurementrangeis0.05-10IU/mLfort-PAactivityand0.1-20ng/mLfort-PAantigen.Theinter-andintra-assayvariationsarelessthan10%and5%,respectively.

KitComposition:

Determination:42samplesinduplicate

1. PLATE+PLATECOVER12x8wellplasticmicrotitrestripsprecoatedwithamonoclonalanti-t-PAcoatingantibodyinbicarbonatebuffer,1%bovineserumalbumin(BSA),(TC-CodeGN).
2. STANDARD1xlyophilizedrecombinantt-PA,calibratedagainstNIBSC86/670(TC-CodeAW).
3. POX-ANTIBODY1xconjugatedantit-PAantibody(concentrated),0,3ml.(TC-CodeKM).
4. INCUBATIONBUFFER1×90ml(PBS;pH7,3)containsstABIliserprotein,0,05%proclinandbluedye,1bottle,,readyforuse(TC-CodeNB)
5. SUBSTRATE–(greencap)1x12mLTMB(tetramethylbenzidine)(TC-CodeKN)readytouse.
6. STOPSOLUTION–(redcap)1x15mLsulphuricacid0.45mol/l(TC-CodeKK)readytouse.
7. WASHINGBUFFER–concentrate(1+11,5)1x80ml(PBSpH7,3)containingdetergent,0,01%merthiolat(TC-CodeNA).
8. PLASMINOGENACTIVATORDETECTIONMIXTURE.1xlyophilizedH-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilidediacetatesalt,GluplasminogenandCyanogenbromidefibrinogenfragments(TCCodeBA).
9. PLASMIN-ACTIVATORDETECTIONMIXTUREDILUTIONBUFFER(whitecap)1x20mL50mMTris,12mMNaCl.Readytouse(TC-CodeDA).

Alsorequired

1. MicroPipettesandamultichannelmicropipette;pipettetips.
2. Glassorplastictesttubesfordilutingthestandard+samples.
3. Laboratorybottlesorbeakersandgraduatedcylindersfordilutingwashandincubationbuffer.
4. Distilledordeionisedwater.
5. Absorbentpapertowels.
6. Microtitreplatewasher(alternatively,washingcanbeperformedmanuallyusingamultichannelpipetteorrepeatingsyringe).
7. Amicrotitrereaderequippedwitha405nmfilterand,ifpossIBLe,a492nmreferencefilter.
8. A37°Cincubator
9. Graphpaper.

MeasurementPriniple:

IntheActibindt-PAtest,anantibody,whichdoesnotinterferewitht-PAfunctionalactivity,iscoatedontoamicrotitreplateandusedtobindt-PAcontainedinplasmatotheplatesurface.

Followinganincubationperiod,non-boundplasmacomponentsarewashedawayandanactivitysubstratesolutioncontainingGlu-plasminogen,CNBr-fragmentsoffibrinogenandachromogenicplasminsubstrateisincubatedintheplate.Theboundt-PAactivatesGlu-plasminogentoyieldplasmin.Thereactionbetweenplasminandthechromogenicplasminsubstratereleasesacolouredproductwhoseconcentrationisproportionaltotheamountofactivet-PAinthetestsample.

Afterphotometricallymeasuringthisreaction,themicrotitreplateiswashedtoremovetheactivitysubstratesolution.Thet-PAantigenremainsboundtotheplate.AhorserADIshperoxidase(POX)conjugatedmonoclonalanti-t-PAantibodywhichrecognizesbothactive,t-PAandinactivet-PAisthenincubatedontheplate.Followingincubationandwashingoftheplate,aPOXsubstrateisusedtoproduceacolouredreactionproductwhoseconcentrationisproportionaltothetotalt-PAcontentofthetestsample.

Background:

Tissueplasminogenactivator(t-PA)isa70,000daltonglycoproteinwhichservesasthemajoractivatorofthebloodfibrinolyticsystem.Itissynthesizedprincipallybyendothelialcellsandreleasedintothebloodstreamfollowingstimulus,e.g.exerciseorvenousocclusion.t-PAisuniqueamongtheplasminogenactivatorsduetothefactthatitsactionishighlyfibrinspecific.Onlyinthepresenceoffibrin,towhichitbinds,doest-PAefficientlyactivateGlu-plasminogentoyieldthecleavageproduct,plasmin.Plasmin,inturn,degradesfibrin,theinsolubleproteinpolymerinthrombi.

tPAcirculatesinplasmaataconcentrationofapproximately2-8ng/mL.However,95%ofcirculatingt-PAiscomplexedwiththeplasminogenactivatorinhibitor,PAI-1,andthusinaninactiveform.Uponvenousocclusiontheconcentrationoft-PAantigenincreasesto15ng/mLorhigherandt-PA’sactivityrisesaccordingly.Thisincreaseisprobablyduetoacombinationofincreasedt-PAreleasebyendothelialcellsandareductionint-PAclearancefromtheocclusionsite.t-PAactivitycanonlybemeasuredinsamplesfromwhichinhibitorsoft-PAhavebeenremoved.IntheActibindsystemthisissimplyaccomplishedbyspecificimmunoabsorptionofthetPAcontainedintheplasmasamplebyanantibodyimmobilizedontoamicrotitreplatesurface.Inthisfirststept-PAandt-PA-PAI-1-complexesareboundtotheplateandactive,non-complexedt-PAistherafterquantifiedusingasolutionofGlu-plasminogen,fibrinogenfragmentsandasyntheticsubstratewhichreactswithplasminformedinthereactiontoyieldacolouredreactionproduct.Lowactivityinthisfirststepcanbeduetoeitheradecreaseinthequantityoftotalt-PAboundtotheplateortoanexcessofPAI-1intheplasmasample,whichinactivatest-PAandleadstotheformationoft-PA-PAI-1-complexes.Todifferentiatebetweenthesetworeasonsfordecreasedt-PAfunction,thesecondpartoftheActibindassayisthenusedtodeterminethetotalamountoft-PAantigenboundontothemicrotiterplatethroughtheuseofaperoxidase-labelledmonoclonalantibody.