BiochemicalEngineeringJournal43(2009)58–63ContentslistsavailableatScienceDirectBiochemicalEngineeringJournaljournalhomepage:www.elsevier.com/locate/bejAhighlysensitiveenzyme-linkedimmunosorbentassayforquantificationofadipocytokinessecretedbymouseadipocytesMinoriMimura1,RisakoNabeshima,MiwakoMaeda,NaofumiShiomi∗DepartmentofBiosphereSciences,KobeCollege,4-1Okadayama,Nishinomiya662-8050,JapanarticleinfoArticlehistory:Received3June2008Receivedinrevisedform24July2008Accepted15August2008Keywords:ELISATNFLeptinAdipocytesabstractThemousepreadipocyte3T3-L1lineisthemostusefulcelllineforthestudyofadipocytes.Adipocytessecreteadipocytokines,andabnormaladipocytokineproductioncancausethemetabolicsyndrome.Althoughitisimportanttounderstandthecharacteristicsofmouseadipocytokinesecretion,itisdif-ficulttoquantifytheseproductsbecausetheyareproducedinlowconcentrations.Here,wedevelopedahighlysensitiveenzyme-linkedimmunosorbentassay(ELISA)fordetectingtheconcentrationsofmouseadipocytokines,suchasTNF�andleptin.Inthismethod,theantigenwassandwichedbyusinggoat-andrabbit-derivedpolyclonalantibodies,andthefluorescenceintensityproducedinthereactionwith4-methylumbelliferyl-�-galacto-sidasepyranosidase(MUG)wasmeasured.TNF�andleptincouldbemeasuredatconcentrationsaslowasapproximately1pg/ml.ByusingourELISAmethod,wealsomeasuredtheconcentrationsofTNF�andleptininmouse3T3-L1preadipocytesandadipocytes.Thedif-ferentiationofpreadipocytesintoadipocytesenhancedTNF�productionandsecretionandreducedtheleptinproduction.TheamountofTNF�producedintheadipocyteswas3.0ng/mg-protein;thisamountwasconsiderablyhigherthanthatofleptin.©2008ElsevierB.V.Allrightsreserved.1.IntroductionThemetabolicsyndrome,adiseasesmanifestedinsomeobesity-inducedconditions,hasbeenremarkedatastandpointofpreservationofalifestyle-relateddiseasesuchasdiabetes.Whiteadiposetissue(WAT)wasinitiallyconsideredtofunctionmerelyasanenergy-storagetissuethataccumulatestriglycerides.How-ever,sincethediscoveryofadipocytokinesinadipocyteproducedbyadipo...
