Signosis/Human TNFα ELISA/EA0203/1 Ea

Description:

TumourNecrosisFactoralpha(TNFa),isaninflammatorycytokineproducedbymacrophages/monocytesduringacuteinflammationandisresponsIBLeforadiverserangeofsignalingeventswithincells,leADIngtonecrosisorapoptosis.Theproteinisalsoimportantforangiogenesisthatiscriticaltothegrowth,progression,andmetastasisofsolidtumors.FurThermore,TNFaisassociatedwithobesity.Itischronicallyelevatedinadiposetissuesofobeserodentsandhumansandmayrepresentanimportantlinkbetweenobesityandinsulinresistance.Inbothobesemiceandhumans,TNFaisoverexpressedinadiposetissue.TNFainhibitsinsulinsignaling,atleastinpartbyblockinginsulinreceptortyrosinekinaseactivityandinducingserinephosphorylationofinsulinreceptorsubstrate-1(IRS-1).However,itisunclearwhatthephysiologicalstimulatorofTNF-aproductionbyadipocyteduringobesityisandhowIRS-1inhibitsthetyrosinekinaseactivityoftheinsulinreceptorafterTNF-atreatmentofthecells.Abetterunderstandingoftheconnection(s)betweentheTNF-aandtheinsulinsignalingpathwayscouldbeimportanttofindacureforthestateofinsulinresistanceobservedduringobesity.

Principle:

TheELISAisbasedontheprincipleofasolidphaseenzyme-linkedimmunosorbentassay.Theassayutilizesamouseanti-humancytokineantibodyforimmobilizationonthemicrotiterwellsandgoatanti-humancytokineantibodiesalongwithstreptavidinconjugatedtohorseradishperoxidase(HRP)fordetection.Thetestsampleisallowedtoreactsimultaneouslywiththetwoantibodies,resultinginthemoleculesofthecytokinebeingsandwichedbetweenthesolidphaseandenzyme-linkedantibodies.Afterincubation,thewellsarewashedtoremoveunbound-labeledantibodies.AHRPsubstrate,TMB,isaddedtoresultinthedevelopmentofabluecolor.ThecolordevelopmentisthenstoppedwiththeadditionofStopSolutionchangingthecolortoyellow.Theconcentrationofthecytokineisdirectlyproportionaltothecolorintensityofthetestsample.Absorbanceismeasuredspectrophotometricallyat450nm.

Data: