Description:
TECO®IntactProinsulinELISAisabioMarkerforadvancedbeta-celldysfunctionandresearchofinsulinresistance.
Proinsulinisproducedinthepancreaticß-cellsandisnormallyfurtherprocessedtoinsulinandC-peptide.Itisonlyseeninlowconcentrationsintheplasmaofnormalsamples.Anincreaseintheinsulindemand,asprovidedbyinsulinresistanceinlaterstagesoftype2diabetesmellitusresearch,canresultinincreasedexpressionofproinsulinintothesample.Theintactmoleculeanditsdegradationproductsareknowntoblockfibrinolysisbecauseofplasminogen-activatorinhibitor(PAI-1)stimulation.
- DiabetesII
- Stagingofinsulinresistanceandb-celldysfunction
- IdentificationofhighrisksubjectsforCAD
- PolycysticovarySyndrome(PCOS)
- Insulinoma
KitComposition:
Reagentsandmaterialssupplied
| Symbol | Description | Format |
| 1 | IntactProinsulinAntibodyCoatedMicrotiterPlate 12stripsof8wells(96breakablewellsintotal), inaframe,Readytouse | 1plate |
| 2 | BlockingBuffer Readytouse | 1x1.5ml |
| 3 | Antibody-HRPConjugate Readytouse | 1x11ml |
| 4 | TMBSubstrate Readytouse | 2x15ml |
| 5 | WashSolution 10timesconcentrated | 1x40ml |
| 6 | StopSolution–0.5MH2SO4 0.5Msulfuricacid,readytouse | 1x15 ml |
| A | StandardA 0pmol/L,lyophilized | 2x3.0ml |
| B | StandardB lyophilized,Concentrationseedatasheet | 1x1.0ml |
| C | StandardC lyophilized,Concentrationseedatasheet | 1x1.0ml |
| D | StandardD lyophilized,Concentrationseedatasheet | 1x1.0ml |
| E | StandardE lyophilized,Concentrationseedatasheet | 1x1.0ml |
| F | StandardF lyophilized,Concentrationseedatasheet | 1x1.0ml |
| L | Control1 lyophilized,Rangeseedatasheet | 1x1.0ml |
| H | Control2 lyophilized,Rangeseedatasheet | 1x1.0ml |
| I | KitInstruction | 1x |
MaterialsRequiredandnotSupplied
- Pipettescapableofdispensing50µl,100µl,150µland300µl
- Graduatedcylindersforreconstitutingordilutingreagents
- Manualaspirationsystemandmulti-channelpipetteorautomaticwasher
- Aquadest
- Vortexmixer
- ELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat450and405nmandwith590-650forreference
- ELISAplateshaker(400rpm)(orbitalshaker)
- Softwarepackagefordatareductionandanalysis
AssayPrinciple&Procedure:
AssayPrinciple
TheTECO®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.
AssayProcedure
Note
Inordertoobtainanoptimaldifferentiationinthecut-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/l
Sensitivity
0.3pmol/l
Specificity
Nocross-reactivityhasbeenobserved:
| HumanInsulin | <10000pmol/l |
| HumanC-Peptide | 50000pmol/l |
| Des(31,32)–Proinsulin | <200pmol/l |
| Split(32,33)–Proinsulin | 5000pmol/l |
| Des(64,65)–Proinsulin* | 200pmol/l |
| Split(65,66)–Proinsulin | 1000pmol/l |
*notpresentinSerumandPlasmasamples
Sample:
Samplevolume
50µl
Sampletype
Serum,EDTA/Heparinplasma,cellculture
Samplepreparation
- Fastingbloodsamplecollection.
- Duetohigherstability,EDTAorheparinplasmasamplesarepreferredtoserumsamples.
- Plasma:thesamplecollectioncantakeplaceinHbA1C-tubes.
- Thesesamplesarestableatroomtemperatureandshouldbecentrifugedwithin48hours.Plasmashouldbeusedintheassayorcanbestoredinaliquots,stable>2yearsat-20°C.
- Serum:centrifugewholebloodwithin4hours.Proteasesdegradeintactproinsulininserum,donotstorelongerthan1dayat2-8°C.
- Serumshouldbeusedintheassayorcanbestoredinaliquotsat-20°C.
- Avoidrepeatedfreeze/thawcycles.
Incubationtime
2.5hours
Species
Human
Referencevalues:
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.
