Description:
DiapharmaPAI-1Antigenisatwo-siteELISAmethodforthemeasurementofPAI-1antigen(Plasminogen-Activator-Inhibitor1).
AssaydesignedwithtwocomplementarymurinemonoclonalantibodiesspecificforPAI-1. Thekitcontainsacalibrator,referencedtotheNIBSCinternationalstandard,andtwocontrolplasmas(lowandhigh).TheassaymeasureshomogeneouslyPAI-1whetheritspresentationis:free,active,latent,inactive,complexedwithtPA,uPAorvitronectin.Controlsareincluded.
KitComposition:
- COAT:MicroELISAplate,containing12stripsof8wells,coatedwithamurinemonoclonalantibodyspecificforhumanPAI-1:Ag,thenstABIlized;theplateispackedinanaluminiumpouchhermeticallysealedinpresenceofadesiccant.
- SD:2vialscontaining50mlofF-SampleDiluent,readytouse.
- STD:3vialsofPAI-1Standard,lyophilized.Whenrestoredwith2mlofF-SampleDiluent,asolutioncontainingabout10ng/mlofrecombinanthumanPAI-1:Agisobtained.TheexactPAI-IAgconcentrationisindicatedontheflyerprovidedinthekit.
- CI:1vialcontaining1mloflyophilizedPlasmaPAI-1ControlIHigh(humanplasma).
- CII:1vialcontaining1mloflyophilizedPlasmaPAI-1ControlIILow(humanplasma).
Note:ThePAI-1:Agconcentrationsandacceptancyrangesforcontrolscanvaryfromlottolot,andareindicatedontheflyerprovidedinthekit. - IC:3vialsofAnti-(h)-PAI-1-HRPimmunoconjugate,amonoclonalantibodycoupledtoHRP,lyophilized.
- CD:1vialof25mlofConjugateDiluent,readytouse.
- WS:1vialof50mlof20foldconcentratedWashSolution.
- TMB:1vialof25mlperoxidasesubstrate:3,3′,5,5′–Tetramethylbenzidinecontaininghydrogenperoxide.Readytouse.
- SA:1vialof6mlof0.45MSulfuricacid(Stopsolution).Readytouse.
Note:Useonlycomponentsfromasamekitlot.Donotmixcomponentsfromdifferentlots,whenrunningtheassay.
MeasurementPrinciple:
First,theimmunoconjugate,whichisamonoclonalantibodyspecificforPAI-1:AgcoupledtohorserADIshperoxidase(HRP),isintroducedintothemicrowellscoatedwithanothermonoclonalantibodyspecificforPAI-1:Ag.Then,thedilutedtestedsampleisimmediatelyintroduced,andtheimmunologicalreactionstarts.Whenpresent,PAI-1:Agbindsontothemonoclonalantibodycoatedsolidphasethroughoneepitope,andfixesthesecondmonoclonalantibodycoupledtoHRPbyanotherepitope.Followingawashingstep,theperoxidasesubstrate,3,3′,5,5′–Tetramethylbenzidine(TMB),inpresenceofhydrogenperoxide(H2O2),isintroducedandabluecolordevelops.WhenthereactionisstoppedwithSulfuricAcid,ayellowcolorisobtained.TheamountofcolordevelopedisdirectlyproportionaltotheconcentrationofhumanPAI-1:Aginthetestedsample.
