Description:
Granulocyte-macrophagecolony-stimulatingfactor(GM-CSF)isacytokinesecretedbymacrophages,Tcells,mastcells,endothelialcellsandfibroblasts.Itfunctionsasawhitebloodcellgrowthfactor.GM-CSFstimulatesstemcellstoproducegranulocytes(neutrophils,eosinophils,andbasophils)andmonocytes.Monocytesexitthecirculationandmigrateintotissue,wheretheymatureintomacrophages.IthasrecentlyshowedthatGM-CSFplaysakeyroleininflammatoryandautoimmunediseases.Principle:
TheELISAisbasedontheprincipleofasolidphaseenzyme-linkedimmunosorbentassay.Theassayutilizesamouseanti-humancytokineantibodyforimmobilizationonthemicrotiterwellsandgoatanti-humancytokineantibodiesalongwithstreptavidinconjugatedtohorserADIshperoxidase(HRP)fordetection.Thetestsampleisallowedtoreactsimultaneouslywiththetwoantibodies,resultinginthemoleculesofthecytokinebeingsandwichedbetweenthesolidphaseandenzyme-linkedantibodies.Afterincubation,thewellsarewashedtoremoveunbound-labeledantibodies.AHRPsubstrate,TMB,isaddedtoresultinthedevelopmentofabluecolor.ThecolordevelopmentisthenstoppedwiththeadditionofStopSolutionchangingthecolortoyellow.Theconcentrationofthecytokineisdirectlyproportionaltothecolorintensityofthetestsample.Absorbanceismeasuredspectrophotometricallyat450nm. |
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