Description:

Technozym®uPACombiActibind®ELISA isanassay kit forthecombinedquantitativedeterminationofconcentrationandactivityofu-PAinhumanplasma.u-PAActibind® assay isbasedonacatchingantibodywhichdoesnotinterferewiththefunctionalactivityofu-PA.Followingthebindingofu-PAinthesamplebytheantibody,functionalactivityofboundu-PAisdeterminedusingGlu-Plasminogenandalowmolecularweightplasminsubstrate.Aftermeasuringthefunctionalactivity,thisdeterminationsystemiswashedawayandboundu-PAantigenisdetectedusingaperoxidase-labeledmonoclonalantiu-PAantibodywhichrecognizesbothfreeu-PAandu-PAinhibitorcomplexes.

Specificity:

TheTechnozym®Actibind®u-PAtestmeasuresu-PAfunctionalactivity,noncomplexedu-PAantigenandu-PA-inhibitorcomplexes.Itisnotaffectedbythepresenceofotherplasminogenactivators.Thetestsystemmeasuresu-PAantigeninarangefrom0-25ng/mlanduPAactivityfrom0-2.5IU/ml.Theinter-andintra-assayvariationsarelessthan10%and5%,respectively.

KitComposition:

1. Plate+PlateCover
   12x8wellplasticmicrotitrestripsprecoatedwithamonoclonalanti-u-PAcoatingantibodyinbicarbonatebuffer,1%bovineserumalbumin(BSA),(TC-CodeGU).
2. Standard
   1xlyophilizedurokinase,calibratedagainstNIBSC87/594(TC-CodeAQ).
3. POX-Antibody
   1xconjugatedpolyclonalantiu-PAantibody(concentrated).(TC-CodeAN).
4. DilutionBuffer –(whitecap)
   1x20ml2.5xconcentrated(PBS,1%BSA,20mMEDTA,10KIU/mlaprotinin,20mMBenzamidine)(TC-CodeDC).
5. POXDilutionBuffer –(whitecap)
   1x12mlPBS,1%BSAReadytouse(TC-CodeDD).
6. Substrate –(greencap)
   1x12mlTMB(Tetramethylbenzidine)insubstratebuffercontainingH2O2.Readytouse(TC-CodeKN).
7. StopSolution –(redcap)
   1x15ml0.5MSulphuricAcid(TC-CodeKK).Readytouse.
8. WashBuffer –(bluecap)
   1x20ml12.5xconcentrated(PBS,0.5%,Tween20)(TC-CodeBE).
9. PlasminogenActivatorDetectionMixture
   1xlyophilizedH-D-norleucyl-hexahydrotyrosyllysine-p-nitroanilidediacetatesaltandGluplasminogen(TCCodeBA).
10. DetectionMixtureDilutionBuffer (whitecap)
    1x20ml50mMTris,12mMNaCl.Readytouse(TC-CodeDA).

MeasurementPrinciple:

IntheActibind®u-PAtestamonoclonalantibodywhichdoesnotblocku-PAfunctionalactivityiscoatedontoamicrotitreplateandusedtobindu-PAcontainedinplasmatotheplatesurface.Followinganincubationperiod,non-boundplasmacomponentsarewashedawayandadetectionmixturecontainingGlu-plasminogenandachromogenicplasminsubstrateisincubatedontheplate.Theboundu-PAactivatesGlu-plasminogentoyieldplasmin.Thereactionbetweenplasminandthechromogenicplasminsubstratereleasesacoloredproductwhoseconcentrationisproportionaltotheamountofactiveu-PAinthetestsample.Afterphotometricallymeasuringthisreaction,themicrotiterplateiswashedtoremovetheactivitysubstratesolution.Theu-PAantigenremainsboundtotheplate.AhorserADIshperoxidase(POX)conjugatedanti-u-PAantibodywhichrecognizesbothactiveu-PAandinactiveu-PAisthenincubatedontheplate.Followingincubationandwashingoftheplate,aPOXsubstrateisusedtoproduceacoloredreactionproductwhoseconcentrationisproportionaltothetotalu-PAcontentofthetestsample.

Background:

Humanurokinase(u-PA)isanenzymewhichfunctionsasanactivatorofthefibrinolyticenzymesystem.ItsABIlitytolysefibrinclotsmakesitusefulasaneffectivethrombolyticagentintheresearch ofavarietyofclinicaldiseasestatesincludingpulmonaryembolismandlocalizedthrombosis.Urokinaseissynthesizedbyandreleasedfromnumerouscelltypesincludingendothelialcells,kidneycellsandmacrophages.Severalmalignanttumors,especiallythoseoftheurogenitalandgastrointestinaltracts,havebeenshowntoproduceincreasedquantitiesofurokinase.

Urokinaseexistsinthreemajorforms:enzymaticallyinactivesinglechainurokinaseorpro-urokinase(scu-PA),enzymaticallyactivetwochainurokinase(tcu-PA)andurokinase-inhibitorcomplexes.scu-PAcirculatesinplasmaataconcentrationof1-2ng/mlandisconvertedtotcu-PAinvivobytheactionofplasminandkallikrein.Eachformofu-PAdisplaysadifferentactivity,differentaffinitiesforGlu-plasminogen,anddifferentratesofinhibitionbyplasmaproteaseinhibitors.

Forresearch admiNISTration,pro-urokinaseisgenerallypreferredoverotherformsofthisplasminogenactivator. Pro-urokinaseisnotinhibitedbyplasminogenactivatorinhibitorsand,althoughnoformofurokinaseishighlyfibrinspecific,pro-urokinaseismorefibrin-orientedthanotherformsofu-PA.Duringthrombolytictesting,pro-urokinaseisconvertedtotheactivetwochainurokinaseandisthensusceptIBLetoinhibition.TheActibind®u-PAassayemploysspecificreagentswhichallowforthemeasurementofurokinaseactivityoriginatingfromboththeactivetwochainformandthesinglechainformofthemolecule.Itdoesnotdetectinactiveurokinase-inhibitorcomplexes.u-PAantigen,however,isdetectedinallthreeforms(scu-PA,tcu-PAandu-PAinhibitorcomplexes)bythisassaysystem.Thisallowsthequantificationofurokinaseinhibitors.

Ifyouwishtodifferentiatebetweenpro-urokinaseandtwo-chainurokinase,theTechnozymscu-PAkitTC11110determinesonlysinglechainurokinase.