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1、浙江農(nóng)業(yè)學報ActaAgriculturaeZhefiangensis,2015,27(5):740—745http://www.~nyxb.en蔣佳穎,王一成,楊孝樸,等.雙抗體夾心ELISA法測定豬僅.干擾素的濃度[J].浙江農(nóng)業(yè)學報,2015,27(5):740—745.DOI:10.3969/j.issn.1004—1524.2015.05.06雙抗體夾心ELISA法測定豬lt-干擾素的濃度蔣佳穎,王一成,,楊孝樸,袁秀芳,李軍星,徐麗華(甘肅農(nóng)業(yè)大學動物醫(yī)學院,甘肅蘭州730070;浙江省農(nóng)業(yè)科學院畜牧獸
2、醫(yī)研究所,浙江杭州310021)摘要:為了能夠準確測定豬血清中豬一干擾素(PoIFN—)含量及豬一干擾素制品的濃度,本研究以制備并經(jīng)過親和層析提純的兔抗豬一干擾素IgG多克隆抗體為包被抗體,以制備的鼠抗豬.干擾素單克隆純化抗體作為二抗,建立了測定豬o【一干擾素濃度的雙抗體夾心ELISA法(DAS—ELISA)。通過條件優(yōu)化,最佳兔抗豬一干擾素IgG多克隆純化抗體包被濃度為12.5Ixg·mL。。,最佳鼠抗豬0【一干擾素單克隆純化抗體作用濃度為6.25txg·mL~,利用本實驗室制備的已知濃度豬0【一干擾素標準蛋白建
3、立標準曲線,確定該方法檢測豬僅。干擾素濃度范圍為0.0781—2.50txg·mL‘。。與Lowry法、凍干稱量法測定豬0【一干擾素濃度相比較,3種方法檢測結(jié)果無顯著差異,但所建立的雙抗體夾心ELISA法能夠檢測血清中豬0【.干擾素濃度,且方法簡便。關鍵詞:豬Ot一干擾素;雙抗體夾心ELISA;定量檢測中圖分類號:S852.4;Q816文獻標志碼:A文章編號:1004—1524(2015)05-0740-06DevelopmentofdoubleantibodysandwichELISAfordetermining
4、theconcentrationofpor—cinea-interferon(PoIFN-)JIANGJia—ying,WANGYi—cheng',YANGXiao—pu’,YUANXiu—fang,LIJun.xing,XULi—hua(AnimalVeterinaryCollege,GansuAgriculturalUniversity,Lanzhou730070,China;eAnimalHta~bandryandVeterinaryInstitute,Zh~jiangAcademyofAgricultura
5、lSciences,Hangzhou310021,China)Abstract:Inordertoaccuratelymeasuretheconcentrationofporcine0【一interferon(PoIFN一僅)inswinesei31mandPoIFN一儀product.DoubleantibodysandwichELISA(DAS-ELISA)methodwasdevelopedbyusingpurifiedrabbitantiPolFN一0【IgGascaptureantibody.a(chǎn)ndpur
6、ifiedmouseantiPoIFN一ⅡIgGasdetectionantibody.TheresultindicatedthatthebestpackageconcentrationofrabbitantiporcinePoIFN一0【IgGwas12.5恤g·mL~,andmouseantiPoIFN—dIgGwas6.25txg·mL~.ThedetectionrangeoftheDAS-ELISAwasbetween0.078l一2.50vg·mLbystandardCHIVeestablishedusi
7、ngtheknownconcentrationofstandardproteinofPoIFN一0【.Comparedwiththelowrymethodandfreeze—dryingweighingmethod,therewasnosignificantdifferencebetweenthethreemethods.AndthisestablishedDAS—ELISAmethodwassimpleandcoulddetectconcentrationofPoIFN·0【inselql/n.Keywords:
8、porcineOt—interferon;doubleantibodysandwichELISA;quantitativedetermination干擾素(Interferon)是一類具有廣譜抗病毒組的調(diào)節(jié)和控制,而且與細胞RNA及蛋白質(zhì)的活性的蛋白質(zhì),其活性的發(fā)揮主要受到細胞基因合成有關,是一種具有抗病毒、調(diào)節(jié)機體免疫功收稿日期:2014.10-2l基金項