2009年6月中国比较医学杂志June,2009第19卷第6期CHINESEJOURNALOFCOMPARATIVEMEDICINEVol.19No.6研究报告七种常见宠物二级抗体的制备和HRP标记林树柱,刘先菊,杨帆,秦川,张连峰(中国医学科学院实验动物研究所北京协和医学院比较医学中心卫生部人类疾病比较医学重点实验室,北京100021)【摘要】目的制备兔抗7种中国家庭中常见宠物的二级抗体,并进行辣根过氧化酶标记,为宠物血清学检测系统的建立提供工具。方法利用饱和硫酸铵沉淀法粗纯抗体后,再利用proteinA或proteinG亲和层析的方法进一步纯化宠物的IgG,免疫大耳白兔制备抗血清,利用免疫双扩散来检测抗血清的效价,并用ProteinA亲和层析的方法来纯化兔抗宠物的二级抗体,采用简易过碘酸钠法对纯化的兔抗宠物的二级抗体进行HRP标记,通过ELISA方法测定标记抗体的效价,并利用Westernblotting考察标记抗体的特异性。结果纯化了狗、家猫、豚鼠、金仓鼠、松鼠、花鼠和龙猫7种宠物的血清IgG,免疫双扩散法测定兔抗这7种宠物的抗血清效价均达到1∶32,并对纯化的兔抗7种宠物的二级抗体进行了HRP标记,ELISA测定标记抗体的效价达到1∶(2000～15000)左右,Westernblots显示标记抗体具有很好的特异性。结论成功制备了HRP标记的兔抗7种中国家庭中常见宠物的二级抗体,为宠物血清学检测体系的建立提供了工具。【关键词】宠物;IgG;兔;辣根过氧化酶【中图分类号】R392【文献标识码】A【文章编号】167127856(2009)0620051204PreparationandHRPLabelingofRabbitPolyclonalAntibodiesagainstSevenPetAnimalsLINShu2zhu,LIUXian2ju,YANGFan,QINChuan,ZHANGLian2feng(KeyLaboratoryofHumanDiseasesComparativeMedicine,MinistryofHealth,InstituteofLaboratoryAnimalScience,ChineseAcademyofMedicalSciences(CAMS)&ComparativeMedicineCentre,PekingUnionMedicalCollege(PUMC),Beijing100021,China)【Abstract】ObjectiveTopreparetherabbitpolyclonalantibodiesagainstsevenpetanimals,whichwerecommonlykeptinChinesefamily,andtolabelthesecondaryantibodieswithhorseradishperoxidasefordevelopingtheserologicaltestsystemofpetanimals.MethodsIgGsofpetanimalswerepurifiedbyprecipitationwithammoniumsulfatecombiningaffinitychromatographyusingProteinAandProteinG.Rabbitanti2serawerepreparedbyrepeatedlyimmunizedrabbitswithpurifiedIgGs.TheantibodiesagainstpetanimalIgGinrabbitseraweredetectedbyagarosedoubleimmunodiffusionandpurifiedusingProteinAaffinitychromatography.TheproductswerelabeledusingHRPbyperiodateoxidationmethod.AndtheactivitiesofthelabeledrabbitpolyclonalantibodiesagainstsevenpetanimalsweredetectedbyELISA.WesternblotswereperformedtoinvestigatethespecificityofHRP2IgGagainsttheIgGofpetanimals.ResultsTheIgGsfromthesevenpetanimalsspecieswerepurified,includingdog,cat,guineapig,goldenhamster,squirrel,westernchipmunk,chinchi