围术期过敏反应.ppt

*Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS)andimmunoblotswereperformedwithcassava,avocado,chestnut,banana,kiwi,andlatexextracts.InSDSPAGE,theextractswereseparatedon12.5%denaturingpolyacrylamidegels.Inimmunoblotexperiments,proteinsweretransferredtoapolyvinylidenefluoridemembrane(HybondP,GEHealthcare,Uppsala,Sweden)accordingtothemethoddescribedbyTowbinetal[12].Themembranewasblockedwithphosphatebufferedsaline(PBS)containing0.1%Tweenfor1houratroomtemperature.Thepatient?sserumwasaddedtothemembraneata1:4dilutioninblockingbufferandleftunderagitationovernightat4oC.Forimmunoblotinhibition,thelatexextractwaspreincubatedwiththepatientsserumfor3hoursatroomtemperature.Themixturewasthenaddedtothemembraneontowhichthecassavaextractproteinshadbeentransferred.Afterseveralwashes,themembranewasincubatedwithhorseradish-peroxidase-conjugatedantihumanIgEantibody(DAKO,Barcelona,Spain)for2hoursatroomtemperature.ImmunodetectionwasperformedusingThecassavaextractcontainedmultipleproteinbands:96.24,83.67,65.52,57.69,48.45,46.44,32.85,29.42,26.97,19.52,17.89,and15.14kd.SDSwasthenperformedwiththeavocado,chestnut,banana,kiwi,andlatexextracts(Figure1).Inaddition,TricineSDSwasperformedinordertoshowthelow-molecular-weightproteinbands.Immunoblottingofthecassavaextractshowedatleast5IgE-bindingbandsof89.75,46.28,26.68,21.38,and19.49kd.Immunoblottingoftheavocadoextractrevealed7IgEbindingbands(42.44,38.06,24.90,22.50,21.00,19.52,and16.83kd),thechestnutextract6bands(44.28,36.37,25.04,21.39,19.55,and17.59kd),thebananaextract3bands(69.33,60.48,and30.10kd),thekiwiextract2bands(33.30and31.50kd),andthelatexextract2IgE-bindingbands(33.30and31.05kd).Immunoblotinhibitionshowed100%inhibitionoftheIgE-bindingpro