Description:TECO®IntactProinsulinELISAisabioMarkerforadvancedbeta-celldysfunctionandresearchofinsulinresistance.Proinsulinisproducedinthepancreaticß-cellsandisnormallyfurtherprocessedtoinsulinandC-peptide.Itisonlyseeninlowconcentrationsintheplasmaofnormalsamples.Anincreaseintheinsulindemand,asprovidedbyinsulinresistanceinlaterstagesoftype2diabetesmellitusresearch,canresultinincreasedexpressionofproinsulinintothesample.Theintactmoleculeanditsdegradationproductsareknowntoblockfibrinolysisbecauseofplasminogen-activatorinhibitor(PAI-1)stimulation.DiabetesIIStagingofinsulinresistanceandb-celldysfunctionIdentificationofhighrisksubjectsforCADPolycysticovarySyndrome(PCOS)InsulinomaKitComposition:ReagentsandmaterialssuppliedSymbolDescriptionFormat 1 IntactProinsulinAntibodyCoatedMicrotiterPlate12stripsof8wells(96breakablewellsintotal), inaframe,Readytouse1plate 2 BlockingBufferReadytouse1x1.5ml 3 Antibody-HRPConjugateReadytouse1x11ml 4 TMBSubstrateReadytouse2x15ml 5 WashSolution10timesconcentrated1x40ml 6 StopSolution–0.5MH2SO40.5Msulfuricacid,readytouse1x15 ml A StandardA0pmol/L,lyophilized2x3.0ml B StandardBlyophilized,Concentrationseedatasheet1x1.0ml C StandardClyophilized,Concentrationseedatasheet1x1.0ml D StandardDlyophilized,Concentrationseedatasheet1x1.0ml E StandardElyophilized,Concentrationseedatasheet1x1.0ml F StandardFlyophilized,Concentrationseedatasheet1x1.0ml L Control1lyophilized,Rangeseedatasheet1x1.0ml H Control2lyophilized,Rangeseedatasheet1x1.0ml I KitInstruction1xMaterialsRequiredandnotSuppliedPipettescapableofdispensing50µl,100µl,150µland300µlGraduatedcylindersforreconstitutingordilutingreagentsManualaspirationsystemandmulti-channelpipetteorautomaticwasherAquadestVortexmixerELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat450and405nmandwith590-650forreferenceELISAplateshaker(400rpm)(orbitalshaker)Softwarepackagefordatareductionandanalysis AssayPrinciple&Procedure:AssayPrincipleTheTECO®humanProinsulinELISAKitisasensitivetwo-sitesandwichenzyme-linkedimmunosorbentassay.Themicrotiterplatesarecoatedwithamonoclonalantibody(S2)specificforanepitopeattheC-peptide/insulinAchainjunction.Theantibodyisabletobindintactproinsulin,des(31,32)-proinsulinandsplit(32,33)-proinsulinbutnotinsulin,C-peptideandtheother“des”and“split”forms.First,ablockingbufferisaddedtotheallocatedwells.Analiquotofpatientsampleisthenaddedtothewells.Afterincubation,thewellsarewashedtoremoveunboundantibodyandotherserumcompounds.Inasecondincubationtime,anenzymelabelledmonoclonalproinsulinantibodyisadded.Thisantibodyisspecificfortheepitopesatinsulinβchain/C-peptidejunction.S53isabletobindtointactproinsulin,des(64,65)-proinsulinandsplit(65,66)-proinsulinbutnotinsulin,C-peptideandother“des”and“split”forms.ThecombinationofthesetwomonoclonalantibodieshastheABIlitytodetectonlytheintacthumanproinsulin.Afterwashing,theremainingoderboundenzymeactivityismeasuredbyaddingachromogenicsubstrate.Theintensityofcolourdevelopmentisproportionaltotheconcentrationofproinsulininthepatientsample.AssayProcedureNoteInordertoobtainanoptimaldifferentiationinthecut-offrange(11pmol/l)itisrecommended tousestandards A till E  (0~60pmol/l)andtomeasuretheabsorptionat450nmwitha referencefilterof590–650nm.Asecondmeasurementofstandards A till  F  (0~100pmol/l) canbedoneat405nmwithareferencefilterof590–650nm.Allowallreagentstostandatroomtemperature(20–25°C)foratleast30minutes.Preparetheframeandtherequirednumberofcoatedstrips  1 .Allocatethewellsofthemicrotiterplateforstandards,controlsandsamples.Pipette50µlofblockingbufferworkingsolution  2  directlyintothebottomofthewells.Pipette50µlofeachstandards A till  F ,controls1and2( L  and  H )andsamplesintothe correspondingwells.Coverthestripsandincubatefor60minutesatroomtemperature(20–25°C)onanorbital shaker(400rpm).Afterincubation,aspiratethewellsbyusingaplatewasherormanuallydecantbyinverting theplate.Washthewells3xwith300mldilutedwashingbuffer.Afterthelastwashcycle taptheinvertedwellsgentlyonadryabsorbentsurfacetoremoveexcesswashsolution.Add100µlofHRPconjugate  3  intothewells.Coverthestripsandincubatefor60minutesatroomtemperature(20–25°C)onanorbital shaker(400rpm).Repeatwashstep5.Pipette150µlofTMBsubstrate  4  intothewellsandincubatefor15–25minutesatroom temperatureonanorbitalshaker(400rpm).Add100µlofstopsolution  6  intothewells,shakefor5secondsonaplateshakerand readtheabsorbancewithin15minutes.Readtheabsorbanceofthewells(450,405nm).Referencefilterat590–650nm.Ifdilutionofsamplesisrequired,dilutionshouldbedonewithzerostandard(recommended dilution1:4).ProtocolsforthedifferentautomaticELISAsystemsareavailable.Range,Sensitivity&Specificity:Range~3–100pmol/lSensitivity0.3pmol/lSpecificityNocross-reactivityhasbeenobserved:HumanInsulin<10000pmol/lHumanC-Peptide50000pmol/lDes(31,32)–Proinsulin<200pmol/lSplit(32,33)–Proinsulin5000pmol/lDes(64,65)–Proinsulin* 200pmol/lSplit(65,66)–Proinsulin1000pmol/l*notpresentinSerumandPlasmasamplesSample:Samplevolume50µlSampletypeSerum,EDTA/Heparinplasma,cellcultureSamplepreparationFastingbloodsamplecollection.Duetohigherstability,EDTAorheparinplasmasamplesarepreferredtoserumsamples.Plasma:thesamplecollectioncantakeplaceinHbA1C-tubes.Thesesamplesarestableatroomtemperatureandshouldbecentrifugedwithin48hours.Plasmashouldbeusedintheassayorcanbestoredinaliquots,stable>2yearsat-20°C.Serum:centrifugewholebloodwithin4hours.Proteasesdegradeintactproinsulininserum,donotstorelongerthan1dayat2-8°C.Serumshouldbeusedintheassayorcanbestoredinaliquotsat-20°C.Avoidrepeatedfreeze/thawcycles.Incubationtime2.5hoursSpeciesHumanReferencevalues:Afterfasting:mean3.99pmol/l+/-1.58SD≤11pmol/l(normalsecretion)>11pmol/l(dysfunctionofsecretion)Background:Proinsulinisproducedinthepancreaticß-cellsandisnormallyfurtherprocessedtoinsulinandC-peptide.Itisonlyseeninlowconcentrationsintheplasmaofhealthysubjects.Anincreaseintheinsulindemand,asprovidedbyinsulinresistanceinlaterstagesoftype2diabetesmellitus,canresultinincreasedexpressionofproinsulinintotheblood.Intactproinsulinisrapidlydegraded,butisconsideredtobeanindependentcardiovascularriskfactor.Theintactmoleculeanditsdegradationproductsareknowntoblockfibrinolysisbecauseofplasminogenactivatorinhibitor(PAI-1)stimulation.Fastingmorningintactproinsulincanbeusedashighlyspecificindicatorofinsulinresistanceandtoresearch theeffectonß-celldysfunction.Subjects withtype2diabetesmellitusandwithelevatedfastingintactproinsulinlevelsshouldberegardedasinsulinresistant.Elevatedfastingintactproinsulinlevelsmayalsobeseeninsubjectswithinsulinoma,abenigninsulinproducingtumorofthepancreas.