Description:

TECO®IntactProinsulinELISAisabioMarkerforadvancedbeta-celldysfunctionandresearchofinsulinresistance.

Proinsulinisproducedinthepancreaticß-cellsandisnormallyfurtherprocessedtoinsulinandC-peptide.Itisonlyseeninlowconcentrationsintheplasmaofnormalsamples.Anincreaseintheinsulindemand,asprovidedbyinsulinresistanceinlaterstagesoftype2diabetesmellitusresearch,canresultinincreasedexpressionofproinsulinintothesample.Theintactmoleculeanditsdegradationproductsareknowntoblockfibrinolysisbecauseofplasminogen-activatorinhibitor(PAI-1)stimulation.

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• 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

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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.

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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.

1. Preparetheframeandtherequirednumberofcoatedstrips  1 .Allocatethewellsofthemicrotiterplateforstandards,controlsandsamples.
2. Pipette50µlofblockingbufferworkingsolution  2  directlyintothebottomofthewells.
3. Pipette50µlofeachstandards A till  F ,controls1and2( L  and  H )andsamplesintothe correspondingwells.
4. Coverthestripsandincubatefor60minutesatroomtemperature(20–25°C)onanorbital shaker(400rpm).
5. Afterincubation,aspiratethewellsbyusingaplatewasherormanuallydecantbyinverting theplate.Washthewells3xwith300mldilutedwashingbuffer.Afterthelastwashcycle taptheinvertedwellsgentlyonadryabsorbentsurfacetoremoveexcesswashsolution.
6. Add100µlofHRPconjugate  3  intothewells.
7. Coverthestripsandincubatefor60minutesatroomtemperature(20–25°C)onanorbital shaker(400rpm).
8. Repeatwashstep5.
9. Pipette150µlofTMBsubstrate  4  intothewellsandincubatefor15–25minutesatroom temperatureonanorbitalshaker(400rpm).
10. Add100µlofstopsolution  6  intothewells,shakefor5secondsonaplateshakerand readtheabsorbancewithin15minutes.
11. Readtheabsorbanceofthewells(450,405nm).Referencefilterat590–650nm.
12. Ifdilutionofsamplesisrequired,dilutionshouldbedonewithzerostandard(recommended dilution1:4).

ProtocolsforthedifferentautomaticELISAsystemsareavailable.

Range,Sensitivity&Specificity:

Range

~3–100pmol/l

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Sensitivity

0.3pmol/l

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Specificity

Nocross-reactivityhasbeenobserved:

Sample:

Samplevolume

50µl

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Sampletype

Serum,EDTA/Heparinplasma,cellculture

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Samplepreparation

• Fastingbloodsamplecollection.
• Duetohigherstability,EDTAorheparinplasmasamplesarepreferredtoserumsamples.
• Plasma:thesamplecollectioncantakeplaceinHbA1C-tubes.
• Thesesamplesarestableatroomtemperatureandshouldbecentrifugedwithin48hours.Plasmashouldbeusedintheassayorcanbestoredinaliquots,stable>2yearsat-20°C.
• Serum:centrifugewholebloodwithin4hours.Proteasesdegradeintactproinsulininserum,donotstorelongerthan1dayat2-8°C.
• Serumshouldbeusedintheassayorcanbestoredinaliquotsat-20°C.
• Avoidrepeatedfreeze/thawcycles.

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Incubationtime

2.5hours

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Species

Human

Referencevalues:

Afterfasting:mean3.99pmol/l+/-1.58SD

Background:

Proinsulinisproducedinthepancreaticß-cellsandisnormallyfurtherprocessedtoinsulinandC-peptide.Itisonlyseeninlowconcentrationsintheplasmaofhealthysubjects.Anincreaseintheinsulindemand,asprovidedbyinsulinresistanceinlaterstagesoftype2diabetesmellitus,canresultinincreasedexpressionofproinsulinintotheblood.Intactproinsulinisrapidlydegraded,butisconsideredtobeanindependentcardiovascularriskfactor.Theintactmoleculeanditsdegradationproductsareknowntoblockfibrinolysisbecauseofplasminogenactivatorinhibitor(PAI-1)stimulation.

Fastingmorningintactproinsulincanbeusedashighlyspecificindicatorofinsulinresistanceandtoresearch theeffectonß-celldysfunction.Subjects withtype2diabetesmellitusandwithelevatedfastingintactproinsulinlevelsshouldberegardedasinsulinresistant.

Elevatedfastingintactproinsulinlevelsmayalsobeseeninsubjectswithinsulinoma,abenigninsulinproducingtumorofthepancreas.

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