Description:
TECO®ADIponectinHighSensitiveELISA.Adiponectinisa30kDaproteinanditspercentageinserumproteinsis0.01%.Invivo,itappearswithdifferentoligomersanditismainlysynthesizedbyadipocytes.Untilnow,IGF-1istheonlyknownnaturalinductorofsynthesis.
Adiponectincanbeuseintheresearchof insulinresistanceandmetabolicsyndromeaswellresearchin increasedriskoftype2diabetesmellitusandcardiovasculardisease.
Today,Adiponectinisthoughttoactasanendogenicinsulinsensitizerbydecreasingexcessiveglucoselevelswithoutincreasinginsulinconcentrationsandbystimulatingtheburningofadiposetissueinmuscleandliver.
Adiponectinisassociatedwithglucoseandlipidmetabolismandisassumedtohavedirectantiatherogeniccharacteristics.
Research significance:
•Obesity
•Atherosclerosis
•Energymetabolism
•Coronaryarterydisease
•Metabolicsyndrome
•Polycysticovarysyndrome(PCOS)
KitComposition:
ReagentsSupplied
| Symbol | Description | Format |
| 1 | AntibodyCoatedWells 12breakapartstripsof8wells(12x8intotal), inaframe,Readytouse | 1plate |
| A | StandardA 2ng/ml–lyophilized | 1x0,75ml |
| B | StandardB 10ng/ml–lyophilized | 1x0,75ml |
| C | StandardC 30ng/ml–lyophilized | 1x0,75ml |
| D | StandardD 70ng/ml–lyophilized | 1x0,75ml |
| E | StandardE 100ng/ml–lyophilized | 1x0,75ml |
| L | Control1 ng/ml–lyophilized,Rangeasindicatedondatasheet | 1x0,5ml |
| H | Control2 ng/ml–lyophilized,Rangeasindicatedondatasheet | 1x0,5ml |
| 2 | DilutionBuffer Readyforuse | 1x125ml |
| 3 | AntibodyHRP-Conjugate Readyforuse | 1x12ml |
| 4 | TMBSubstrate Readyforuse | 1x12ml |
| 4 | WashBuffer 20timesconcentrated | 1x50ml |
| 6 | StopSolution–0,2MH2S04 0,2Msulfuricacid,readyforuse | 1x12 ml |
| 7 | CoverforMicrotiterplate,adhesive | 2pieces |
| I | Kitinstruction | 1x |
Materialsrequiredbutnotsupplied
- Pipettescapableofdispensing5µl,40µl,80µL,100µl,320µl,350µl,500µl,560µland750µl
- Graduatedcylindersforreconstitutingordilutingreagents
- ManualAspirationSystemandmulti-channelpipetteorautomaticwasher
- Aquadest
- Vortexmixer
- ELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat450and405nm(Reference:590–650nm)
- ELISAplateshaker(≥400rpm)(orbitalshaker)
- Softwarepackagefordatagenerationandanalysis
AssayPrinciple&Procedure:
AssayPrinciple
TheTECO®assaykitforAdiponectinisaso-calledSandwich-Assayusingtwospecificandhighaffinitymonoclonalantibodies.TheAdiponectininthesamplesbindstothefirstantibodycoatedonthemicrotiterplate.InthefollowingstepthesecondspecificantiAdiponectin-antibodybindsinturntotheimmobilizedAdiponectin.Thesecondantibodyisbiotinylatedandwillbeincubatedinamixturewithastreptavidin-peroxidase-enzymeconjugate.IntheclosingsubstratereactiontheturnofthecolorwillbecatalyzedquantitativelydependingontheAdiponectin-levelofthesamples.
AssayPrinciple
Alldeterminations(standards,dilutedserumcontrolsanddilutedsamples)shouldbeassayed induplicate.Whenperformingtheassay,thestandards,controlseraandsamplesshouldbepipetted asfastaspossIBLe(<15minutes).
Toavoiddistortionsduetodifferencesinincubationtimes,HRPconjugate,substratesolutionand stopsolutionshouldbeaddedtotheplateinthesameorderandwiththesametimeintervalas thesamples.
Allowallreagentstostandatroomtemperature(20–25°C)foratleast30minutes.Afterreconstitution, keepthereagentsatroomtemperaturefor15minutesandthenmixgentlybeforeuse.
- Preparetheframeandtherequirednumberofcoatedstrips 1.
- Allocatethewellsofthemicrotiterplateforstandards,controlsandsamples.Dilutesamples(seekitinstructionspage11)of andcontrols(1:300).
- Pipette100µldilutionbuffer 2 induplicateintowells(blank).
- Pipette100µlofeachstandards( A till E ),dilutedControlsera( L and H )andsamples (seedilutionprotocol)intothecorrespondingwells.
- Coverthewellswithsealingtapeandincubatetheplatefor1houratroomtemperature (20–25°C)onaplateshaker(≥400–500rpm).
- Afterincubation,aspiratethewellsbyusingaplatewasherormanuallydecantbyinverting theplate.Washthewells3xwith350mldilutedwashingbuffer(15secondsincubationper cycle).Afterthelastwashcycletaptheinvertedwellsgentlyonadryabsorbentsurfaceto removeexcesswashsolution.
- Pipette100µloftheantibodyHRPconjugate 3 ineachwell.
- Coverthewellswithsealingtapeandincubatetheplatefor30minutesatroomtemperature (20–25°C)onaplateshaker(≥400–500rpm).
- Washthewells3timeswithwashingbufferasdescribedinstep6.
- Pipette100µloftheTMBsubstratesolution 4 ineachwell.
- Incubatetheplatefor15minutes,inthedark,atroomtemperature(20–25°C).
- Add100µlofstopsolution E ineachwell.
- Measurethecolorreactionwithin30minutesat450nm(referencefilterbetween 590–650nm).Withstrongcolorreactione.g.>3ODalsomeasureat405nm.
ProtocolsforthedifferentautomaticELISAsystemsareavailable.
Range&Sensitivity:
Range
1–100ng/mlnativeAdiponectin
Sensitivity
<0.6ng/ml
Sample:
Samplevolume
5µl(dilute>1:300serumandplasma).ForotherBIOLOGicalfluidsseeprotocol.
Sampletype
Serum,heparinplasma,breastmilk,urine,saliva,CSF,cellculture
Samplepreparation
- Bloodcollection–fastingisrecommended.
- Samplesarestableformaximum2daysatroomtemperature.Long-termstoragestableformaximum2yearsat-20°C.
- Max.5freezeandthawcycles.
Incubationtime
2hours
Species
Human
Referencevalues:
| mg/l | |
| MenAdult | 8-10 |
| FemaleAdult | 10-12 |
| cut-off | 10 |
Comprehensivereferencedatarelatedtoageandgenderareavailableforthistest.
Background:
Adiponectinisa30kDaprotein,presenting0,01%ofserumproteins.Itismainlysynthesized byadipocytes,butmusclecellsandhepatocyteshavealsotheABIlitytosynthesizeAdiponectin. Untilnow,IGF-Iistheonlyknownnaturalinductorofthesynthesis.ItconsistsofaCollagenlike N-terminalandaglobularC-terminaldomain.InvivoAdiponectinappearswithdifferent oligomers.Besidethetrimerandditrimer,highmolecularmultimersalsoexist.Uptonow twodifferentreceptorsareknown,bothreceptorsareubiquitaryexpressed,thoughthe distributioninthetissuesvaries.TheAdiponectinReceptor1(AdipoR1)issynthesized especiallyinmuscle-andAdipoR2inlivertissue.
Aspecialsignificanceforhighmolecularmultimers,asdescribedrepeatedly,could notbeproveninacomparativestudyofthreetestsystems.
