Description:

Technozym®ProteinCINHActibind® isan ELISAkitfor determinationofactiveProteinCInhibitorantigen.Theassayisbasedontheimmobilizationoffunctionallyactiveurokinasetoplatesbymeansofamonoclonalantibody.ProteinCInhibitorcontainedinthetestsamplebindstou-PAandisthenquantifiedusingaperoxidase-labeledmonoclonalanti-ProteinCInhibitorantibody.TheActibind®PCIcanbeusedasaquantitativeassayforthedeterminationofactive antigenlevelsofPCIinsubjectswithdisseminatedintravascularcoagulationandin atherioscleroticresearch.

KitComposition:

Determinations:42samplesinduplicate

1. MicrotitrePlate
   12x8wellplasticmicrotitrestripsprecoatedwithmonoclonalantiurokinase coatingantibody.
2. Standard
   1xlyophilizedpooledhumanplasma
3. Urokinase
   1xlyophilizedurokinase800U/vial
4. POX-Antibody
   1xconjugatedmonoclonalantiPCIantibody(concentrated),bluecolor
5. SampleDilutionBuffer –(whitecap)
   2x20ml2.5-foldconcentratedPBSwithBSA
6. POXDilutionBuffer –(whitecap)
   1x12mLPBSwithBSA.
7. Substrate –(greencap)
   1x12mlTMB(Tetramethylbenzidine)insubstratebuffercontainingH2O2. Readytouse.
8. StopSolution –(redcap)
   1x15ml0.5MSulphuricAcid.Readytouse.
9. WashBuffer –(bluecap)
   1x20ml12.5-foldconcentratedPBSwithTween20
10. PCIActibind®HighControl
    1xlyophilizedpooledhumanplasmaforlot-specificconcentrationseelabelon vial

AdditionalMaterials (requiredbutnotsupplied)

1. MicroPipettesandamultichannelmicropipetteormultistepperpipette, pipettetips
2. Glassorplastictesttubesfordilutingthesamples.
3. Laboratorybottlesorbeakersandgraduatedcylindersfordilutingwash anddilutionbuffer
4. Distilledordeionisedwater
5. Absorbentpapertowels
6. Microtitreplatewasher(alternatively,washingcanbeperformed manuallyusingamultichannelpipette)
7. Microtitrereaderequippedwitha450nmfilterand,ifpossIBLe,a620nm referencefilter
8. Incubator(37°C)

MeasurementPrinciple:

TheTechnozym®Actibind®PCItestisasolidphaseenzymeimmunoassayinwhichananti-u-PA monoclonalantibodythatdoesnotinterferewiththeactivesiteontheurokinase antigenmoleculeiscoatedontoaplasticmicrotitreplate.Urokinaseisincubatedon theplateleavingitsactivesiteaccesibleforcomplexformationwithactivePCI containedinthesample.Followingthebindingofthesampletheplateiswashedand enzyme-labelled(POX=horserADIshperoxidase)monoclonalanti-PCIwhich recognizesanothersiteontheactivePCIantigenisincubatedontheplate.The quantityofPOXwhichbindsisproportionaltothequantityofactivePCIantigeni.e. non-complexedantigencontainedintestsamples.UnboundPOX-Abiswashedaway andasubstratewhichreactswiththeperoxidaseenzymeisadded,leadingtoacolor changeproportionaltotheamountofenzymebound.Theenzymereactionisstopped afteraspecificincubationtime.Theabsorbancesofthewellsarethenmeasuredand thevaluesobtainedareusedtoconstructastandardcurvefromwhichsamplevalues canbeextrapolated.

StandardCurve:

Background:

ProteinCInhibitor(PCI)isamemberoftheserineproteaseinhibitor(Serpin) superfamilywithhomologytoalpha-1-antichymotrypsin,alpha-1-antitrypsin, antithrombinIII,ovalbuminandangiotensinogen.PCIwithanapparentmolecular weightof57KDhasbeendescribedinavarietyofBIOLOGicalfluidsincludingblood plasma(4µg/ml),urine(250ng/ml)andseminalplasma(200µg/ml). Glycosaminoglycan(GAG)dependentPCIinhibitsactivatedproteinC(APC),twochain urokinase(u-PA),two-chaintissueplasminogenactivator(t-PA),thrombin,factor Xa,andfactorXIainreactionsstimulatedbyheparin.Howeverithasrecentlybeen shownthatthePCI-tissuekallikreininteractionisinhibitedbyheparin. GlycosaminogylcansmaythereforeregulatetheenzymespecificityofPCI. PCIinhibitsitstargetproteasesbyformingSDSstable1:1complexes.Uponcomplex formationthereactivesitepeptidebondoftheinhibitoriscleavedbytheproteaseand thecarboxy-terminalpeptideisreleasedfromtheinhibitor.Dependingonthetarget protease,complexesdissociatemoreorlessslowlyandcleavedinactivePCI (Mr=54,000)andactiveenzymearereleased.

AlthoughPCIhasbeenshowntoinhibitseveralenzymes,itsprecisephysiologicalrole hasyettobedefined.However,thefactthatPCIhasbeendeterminedinhigh concentrationsinavarietyofbiologicalfluidsindicatesitsimportanceasa physiologicalserineproteaseinhibitorwhetheritbeasaninhibitorofaspecificserine proteaseinvivoorasageneralinhibitorfunctioningtoprotecttissuesfromprotease action.

LowvaluesofbothProteinCantigenandfunctionalactivityofPCIhavebeen determinedinsubjects withdisseminatedintravascularcoagulationandlowantigen valuesinliverdiseaseandhighPCIactivityinsubjects ofMI.uPA-PCIcomplexesare formedinsubjectsundergoingthrombolytictesting withu-PAwhentheconcentration ofu-PAexceedstheinhibitorycapacityofplasminogenactivatorinhibitor-1(PAI-1), theprimaryinhibitorofu-PAinvivo.