Description:

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

Leptincanbeusedintheresearchof reproductionandanumberofmetabolicandendocrineaxes.

Duetoitspleiotropiceffects,leptinisavaluableresearchtoolwithregardto:

•Metabolicsyndromestudies
•Adiposityresearch
•Cachexiaandothermetabolicstudies
•Eatingdisorderresearch

KitComposition:

ReagentsSupplied

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.

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

Sensitivity

LLOD<0.25ng/ml
LLOQ1ng/ml

Sample:

Incubationtime

2hours

Samplevolume

20µl

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.

Diapharma Group，Inc

Diapharma Group INC成立于1997年，研究人员具有多年临床以及研发经验，专注于血液病领域的试剂盒，试剂以及仪器，主要涵盖血栓形成、止血、血小板功能分析、细胞凋亡、生态毒理学等。

The Diapharma Group, Inc. formed on January 1, 1997 from Pharmacia Hepar, Inc. in Franklin, Ohio, and began as the exclusive US and Canadian distributor of the Chromogenix substrates and assays.

Over a quarter-century ago, Chromogenix developed the first chromogenic substrate technology under the former name, Kabi Diagnostica. Kabi later merged with Pharmacia. Some of Diapharma’s current staff worked inside the Chromogenix department within Pharmacia’s heparin manufacturing plant.

In 1998, Diapharma moved to West Chester, Ohio, where it remains today. Over the years, Diapharma expanded its product line to include a variety of hemostasis, cell death, platelet function, ecotoxicology, assays, reagents, antibodies and instruments from superior manufacturers.

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