
Description:
Humanleptinisaproteinof167aminoacidsthatplaysakeyroleinregulatingenergyintakeandenergyexpenditure,includingtheregulation(decrease)ofappetiteand(increase)ofmetabolism.LeptinisproducedbyADIposetissueandthelevelofcirculatingleptinisdirectlyproportionaltothetotalamountoffatinthebody.OnceleptinhasboundtotheOb-Rbreceptor,itactivatesStat3,whichisphosphorylatedandtravelstothenucleuswhereitmediatesgeneexpression.Oneofthemaineffectsongeneexpressionisthedown-regulationoftheexpressionofendocannABInoids,responsIBLe—amongtheirmanyotherfunctions—forincreasingappetite.Thereareotherintracellularpathwaysactivatedbyleptin,butlessisknownabouthowtheyfunctioninthissystem.Althoughleptinisacirculatingsignalthatreducesappetite,ingeneral,obesepeoplewhoareresistanttotheeffectsofleptinhaveanunusuallyhighcirculatingconcentrationofleptin,inthesamewaythatpeoplewithtype2diabetesareresistanttotheeffectsofinsulin.Leptinisalsofoundtostimulateendothelialcellproliferationandangiogenesis.Principle:
TheELISAisbasedontheprincipleofasolidphaseenzyme-linkedimmunosorbentassay.Theassayutilizesamouseanti-humancytokineantibodyforimmobilizationonthemicrotiterwellsandgoatanti-humancytokineantibodiesalongwithstreptavidinconjugatedtohorseradishperoxidase(HRP)fordetection.Thetestsampleisallowedtoreactsimultaneouslywiththetwoantibodies,resultinginthemoleculesofthecytokinebeingsandwichedbetweenthesolidphaseandenzyme-linkedantibodies.Afterincubation,thewellsarewashedtoremoveunbound-labeledantibodies.AHRPsubstrate,TMB,isaddedtoresultinthedevelopmentofabluecolor.ThecolordevelopmentisthenstoppedwiththeadditionofStopSolutionchangingthecolortoyellow.Theconcentrationofthecytokineisdirectlyproportionaltothecolorintensityofthetestsample.Absorbanceismeasuredspectrophotometricallyat450nm. |
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