Description:
TECO®Leptin.Leptin,theproductoftheobgene,isarecentlydiscoveredproteohormone.ItisalmostexclusivelyproducedbydifferentiatedADIpocytesandisthoughttoplayakeyroleintheregulationofbodyweight.Leptinhasaninfluenceonthecentralnervoussystem,mainlyonthehypothalamus,bysuppressingfoodingestionandincreasingenergyconsumption.
Leptincanbeusedintheresearchof reproductionandanumberofmetabolicandendocrineaxes.
Duetoitspleiotropiceffects,leptinisavaluableresearchtoolwithregardto:
•Metabolicsyndromestudies
•Adiposityresearch
•Cachexiaandothermetabolicstudies
•Eatingdisorderresearch
KitComposition:
ReagentsSupplied
| Symbol | Description | Format |
| 1 | AntibodyCoatedWells 12breakapartstripsof8wells(12x8intotal), inaframe,Readytouse | 1plate |
| A | StandardA 1ng/ml–lyophilized | 1x0.75ml |
| B | StandardB 10ng/ml–lyophilized | 1x0.75ml |
| C | StandardC 25ng/ml–lyophilized | 1x0.75ml |
| D | StandardD 50ng/ml–lyophilized | 1x0.75ml |
| E | StandardE 100ng/ml–lyophilized | 1x0.75ml |
| L | Control1 lyophilized,Rangeasindicatedondatasheet | 1x0.5ml |
| H | Control2 lyophilized,Rangeasindicatedondatasheet | 1x0.5ml |
| 2 | DilutionBuffer Readyforuse | 1x25ml |
| 3 | Antibody-HRP-Conjugate Readyforuse | 1x12ml |
| 4 | TMBSubstrate Readyforuse | 1x12ml |
| 5 | WashBuffer 20timesconcentrated | 1x50ml |
| 6 | StopSolution–0.2MH2S04 0.2Msulfuricacid,readyforuse | 1x12ml |
| 7 | CoverforMicrotiterplate,adhesive | 2pieces |
| I | Kitinstruction | 1x |
MaterialsRequiredandnotSupplied
- Pipettescapableofdispensing20µl,100µl,350µl,500µland750µl
- Graduatedcylindersforreconstitutingordilutingreagents
- Manualaspirationsystemandmulti-channelpipetteorautomaticwasherforELISAplates
- Aquadest
- Vortexmixer
- ELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat450nm(Reference:590–650nm).
- ELISAplateshaker(≥350rpm)(orbitalshaker)
- Softwarepackagefordatagenerationandanalysis
AssayPrinciple&Procedure:
AssayPrinciple
TheTECO®assaykitforhumanleptinisaso-calledsandwich-assayusingtwospecificandhighaffinitymonoclonalantibodies.Theleptininthesamplesbindstothefirstantibodycoatedonthemicrotiterplate.Inthefollowingstepthesecondanti-Leptin-Antibodybindsinturntotheimmobilizedleptin.Thesecondantibodyisbiotinylatedandwillbeincubatedinamixturewithastreptavidin-peroxidase-enzymeconjugate.Intheclosingsubstratereactiontheturnofthecolorwillbecatalyzedquantitativelydependingontheleptin-levelofthesamples.
AssayProcedure
Alldeterminations(standards,controlseraandsamples)shouldbeassayedinduplicate.When performingtheassay,thestandards,controlseraandthesamplesshouldbepipettedasfast aspossIBLe(<15minutes).
Toavoiddistortionsduetodifferencesinincubationtimes,HRPconjugate,substratesolution andstopsolutionshouldbeaddedtotheplateinthesameorderandwiththesametime intervalasthesamples.
Allowaallreagentstostandatroomtemperature(20–25°C)foratleast30minutes.
- Preparetheframeandtherequirednumberofcoatedstrips 1 .
- Allocatethewellsofthemicrotiterplateforstandards,controlsandsamples.
- Pipette100µldilutionbuffer 2 intoallwells.
- Add20µldilutionbufferinduplicate(Blank).
- Pipette20µlofeachstandard( A till E ),controlsera( L and H )andsamplesintothe correspondingwells.
- Coverthewellswithsealingtapeandincubatetheplatefor1houratroomtemperature onaplateshaker(≥350rpm).
- Afterincubation,aspiratethewellsbyusingaplatewasherormanuallydecantbyinverting theplate.Washthewells3xwith350µldilutedwashingbuffer(15secondsincubationper cycle).Afterthelastwashcycletaptheinvertedwellsgentlyonadryabsorbentsurfaceto removeexcesswashsolution.
- Pipette100µloftheantibodyHRPconjugateineachwell.
- Coverthewellswithsealingtapeandincubatetheplatefor30minutesatroomtemperature onaplateshaker(≥350rpm).
- Afterincubationwashthewells3timeswithwashingbufferasdescribedinstep7.
- Pipette100µloftheTMBsubstratesolution 4 ineachwell.
- Incubatetheplatefor15minutes,inthedark,atroomtemperature(20–25°C).
- Add100µlofstopsolution 6 ineachwell.
- Measurethecolorreactionwithin30minutesat450nm(referencefilterbetween 590–650nm).Withstrongcolorreactione.g.>3OD,alsomeasureat405nm.
ProtocolsforthedifferentautomaticELISAsystemsareavailable.
Range&Sensitivity:
Range
1–100ng/ml,recombinantleptinWHONIBSC97/594
Sensitivity
LLOD<0.25ng/ml
LLOQ1ng/ml
Sample:
Incubationtime
2hours
Samplevolume
20µl
Sampletype
Serum,heparinandEDTAplasma,urine,saliva,cellculture.
Samplepreparation
- Normalfoodintakerhythmprovided,samplesshouldbecollectedtill2p.m.Leptinshowsamoderatecircadianvariationwithapeakat2a.m.,theleptinvaluesatthattimeareabout30to100%higher.
- Thisvariationtogetherwiththeinfluenceoffoodintakeneedstobetakenintoaccountwhenbloodsamplesarecollected.Wholebloodshouldberefrigeratedassoonaspossiblefollowingcollection.
- Samplesarestableformaximal2daysatroomtemperature.
- Long-termstoragestableformaximal2yearsat-20°C.
- Max.5freezeandthawcycles.
Species
Human
Referencevalues:
Leptinlevelsdependonageandgenderandmustbereferredtothepercentagebodyfat(suchasBMI).
Comprehensiveclinicalreferencedataareavailableforthistest.
Background:
Leptin,theproductoftheobgeneisarecentlydiscovered(1994)single-chainproteohormone withamolecularweightof16kD,whichisthoughttoplayakeyroleintheregulation ofbodyweight.Itsaminoacidsequenceexhibitsnomajorhomologieswithotherproteins. Leptinisalmostexclusivelyproducedbydifferentiatedadipocytes.Itactsonthecentral nervoussystem,inparticularthehypothalamus,therebysuppressingfoodintakeandstimulating energyexpenditure.Leptinreceptors–alternativelysplicedformsexistthatdiffer inlength–belongtothecytokineclassIreceptorfamily.Theyarefoundubiquitouslyin thebodyindicatingageneralroleofleptin,whichiscurrentlynotfullyunderstood.
Acirculatingformoftheleptinreceptorexistswhichactsasoneofseveralleptinbinding proteins.Besidesitsmetaboliceffects,leptinwasshowntohaveastronginfluenceona numberofendocrineaxes.Inmalemice,itbluntedthestarvation-inducedmarkeddeclineofLH, testosterone,thyroxineandtheincreaseofACTHandcorticosterone.Infemalemice,leptin preventedthestarvation-induceddelayinovulation.Ob/obmice,whichareleptindeficient duetoanobgenemutation,areinfertile.ThisdefectcouldbecorrectedbyadmiNISTrationof leptin,butnotthroughweightlossduetofasting,suggestingthatleptinispivotalfor reproductivefunctions.
Alltheseactionsmay,atleastinpart,beexplainedbythesuppressiveeffectofleptinon neuropeptideY(NPY)expressionandsecretionbyneuronsinthearcuatenucleus. NPYisastrongstimulatorofappetiteandisknowntobeinvolvedintheregulationof variouspituitaryhormones,e.g.suppressionofGHthroughstimulationofsomatostatin, suppressionofgonadotropinsorstimulationofthepituitary-adrenalaxis.
Themostimportantvariablethatdeterminescirculatingleptinlevelsisbodyfatmass. Obviously,underconditionsofregulareatingcycles,leptinreflectstheproportionofadipose tissueshowinganexponentialrelationship.Thisconstitutivesynthesisofleptinis modulatedbyanumberofnon-hormonalandhormonalvariables.Stimulatorsinbothrodents andhumansareoverfeeding,insulinandglucocorticoids. Suppressionhasbeenshownforfasting,cAMPandbeta-3-adrenoceptoragonists. Fromthesefindingsitbecomesclearthatleptinisanintegralcomponentof researchwithin metabolic andendocrinefeedbackloops.
