Description:

DiapharmaPAI-1Activityisabio-immunoassayforthemeasurementofPAI-1activity.

AssaydesignedwithanELISAplatecoatedwithactivetPA,thenstABIlised,whichbindsactivePAI-1presentinplasmaorintestedsample.Thislatter,isthenrevealedwithaspecificmurinemonoclonalantibody,specificforPAI-1andlabelledwithHorseRADIshPeroxidase(HRP).

Thekitcontainsacalibrator,referencedtotheNIBSCinternationalstandard,andtwocontrolplasmas(lowandhigh).ThisassayisspecificforPAI-1activity.Controlsareincluded.

KitComposition:

• COAT:MicroELISAplate,containing12stripsof8wells,coatedwithtPA,thenstabilized;theplateispackedinanaluminiumpouchhermeticallysealedinpresenceofadesiccant.
• SD:2vialscontaining50mlofF-SampleDiluent,readytouse.
• Cal:3vialsofPAI-1ActivityCalibrator,lyophilized.Eachvial,whenrestoredwith2mlofF-SampleDiluent,allowsobtainingthecalibrator.TheexactPAI-1Activityconcentrationisindicatedontheflyerprovidedinthekit(about10ng/ml).
• CI:1vialcontaining1mloflyophilizedPlasmaPAI-1ControlIHigh(humanplasma).
• CII:1vialcontaining1mloflyophilizedPlasmaPAI-1ControlIILow(humanplasma).
  Note:ThePAI-1ActivityandacceptancyrangesforcontrolsIandIIcanvaryfromlottolot,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:Useonlycomponentsfromkitswiththesamelotnumber.Donotmixcomponentsfromdifferentlotsofkitswhenrunningtheassay.

MeasurementPrinciple:

ThedilutedtestedplasmaorBIOLOGicalfluidisintroducedintoamicrowellcoatedwithrecombinanttPA.Whenpresent,PAI-1bindstocoatedtPA.OnlytheactivePAI-1reactswithtPAandisfixedonthesolidphase.Followingawashingstep,theimmunoconjugate,whichisamousemonoclonalantibodyspecificforhumanPAI-1andcoupledtohorseradishperoxidase(HRP),isintroduced,andbindstoitsspecificepitopeonimmobilizedPAI-1.Followinganewwashingstep,theperoxidasesubstrate,Tetramethylbenzidine(TMB)inpresenceofhydrogenperoxide(H2O2),isintroducedandabluecolordevelops.ThecolorturnsyellowwhenthereactionisstoppedwithSulfuricAcid.TheamountofcolordevelopedisdirectlyproportionaltotheamountofhumanPAI-1Activityinthetestedsample.