Description:

TECO®Leptin.Leptin,theproductoftheobgene,isarecentlydiscoveredproteohormone.ItisalmostexclusivelyproducedbydifferentiatedADIpocytesandisthoughttoplayakeyroleintheregulationofbodyweight.Leptinhasaninfluenceonthecentralnervoussystem,mainlyonthehypothalamus,bysuppressingfoodingestionandincreasingenergyconsumption.

Leptincanbeusedintheresearchof reproductionandanumberofmetabolicandendocrineaxes.

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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

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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.

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AssayProcedure

Alldeterminations(standards,controlseraandsamples)shouldbeassayedinduplicate.When performingtheassay,thestandards,controlseraandthesamplesshouldbepipettedasfast aspossIBLe(<15minutes).

Toavoiddistortionsduetodifferencesinincubationtimes,HRPconjugate,substratesolution andstopsolutionshouldbeaddedtotheplateinthesameorderandwiththesametime intervalasthesamples.

Allowaallreagentstostandatroomtemperature(20–25°C)foratleast30minutes.

1. Preparetheframeandtherequirednumberofcoatedstrips 1 .
2. Allocatethewellsofthemicrotiterplateforstandards,controlsandsamples.
3. Pipette100µldilutionbuffer 2  intoallwells.
4. Add20µldilutionbufferinduplicate(Blank).
5. Pipette20µlofeachstandard( A till E ),controlsera( L and H )andsamplesintothe correspondingwells.
6. Coverthewellswithsealingtapeandincubatetheplatefor1houratroomtemperature onaplateshaker(≥350rpm).
7. Afterincubation,aspiratethewellsbyusingaplatewasherormanuallydecantbyinverting theplate.Washthewells3xwith350µldilutedwashingbuffer(15secondsincubationper cycle).Afterthelastwashcycletaptheinvertedwellsgentlyonadryabsorbentsurfaceto removeexcesswashsolution.
8. Pipette100µloftheantibodyHRPconjugateineachwell.
9. Coverthewellswithsealingtapeandincubatetheplatefor30minutesatroomtemperature onaplateshaker(≥350rpm).
10. Afterincubationwashthewells3timeswithwashingbufferasdescribedinstep7.
11. Pipette100µloftheTMBsubstratesolution 4  ineachwell.
12. Incubatetheplatefor15minutes,inthedark,atroomtemperature(20–25°C).
13. Add100µlofstopsolution  6 ineachwell.
14. Measurethecolorreactionwithin30minutesat450nm(referencefilterbetween 590–650nm).Withstrongcolorreactione.g.>3OD,alsomeasureat405nm.

ProtocolsforthedifferentautomaticELISAsystemsareavailable.

Range&Sensitivity:

Range

1–100ng/ml,recombinantleptinWHONIBSC97/594

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Sensitivity

LLOD<0.25ng/ml
LLOQ1ng/ml

Sample:

Incubationtime

2hours

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Samplevolume

20µl

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Sampletype

Referencevalues:

Leptinlevelsdependonageandgenderandmustbereferredtothepercentagebodyfat(suchasBMI).

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.

 

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