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时间:2020-05-15
《黄瓜细菌性白枯病菌特异性抗体制备及其ELISA检测方法的建立.pdf》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
1、2011年21(2)生物技术57黄瓜细菌性白枯病菌特异性抗体制备及其ELISA检测方法的建立孙艳秋,赵奎华“,刘长远,曹远银(1.沈阳农业大学植物保护学院,辽宁沈阳l10866;2.辽宁省农业科学院植物保护研究所,辽宁沈阳110161)摘要:目的:制备黄瓜细菌性白枯病菌特异性抗体,建立其酶联免疫吸附分析(ELISA)的检测方法。方法:分别以黄瓜细菌性白枯病菌胞内蛋白和菌体为免疫原,制备两种抗体,优化间接ELISA检测条件。结果:间接ELISA法测定两种纯化的菌体抗体(J4)和胞内蛋白抗体(P2)效价分别为1:32000和1:4000,二抗的最佳稀释度为l:3000,方阵试验表明J
2、4的工作浓度为1:16000,抗原的最佳包被浓度为10cfu/ml,P2的工作浓度为1:2000,抗原的最佳包被浓度为10cfu/ml,两种抗体的灵敏度均为10cfu/ml,与黄瓜细菌性角斑病菌等26个菌株无交叉反应,特异性强。结论:成功制备了黄瓜细菌性白枯病菌特异性抗体,建立了ELISA检测方法。关键词:黄瓜细菌性白枯病菌;抗体制备;效价;酶联免疫吸附分析;检测中图分类号:$432.42文献标识码:Adoi:10.3969/j.issn.1004—311X.2011.02.049PreparationofSpecificAntibodiesandDevelopmentofInd
3、irectELISAfortheDetecti0n0fPseudomonasviridOTavaonCucumberSUNYan—qiui1,ZHA0Kui—hua’.LIUChang—yuan。CA0Yuan—yin(1.CollegeofPlantProtection,ShenyangAgriculturalUniversity,Shenymag110866,China;2.InstituteofPlantProtection,LiaoningAcademyofAgriculturalSciences,Shenyangl10161,China)Abstract:Objecti
4、ve:ThespecificpolyclonalantibodiesofPseudomonasvindiflavaoncucumberwereprepared,andELISAmethodwases-tablished.Method:TwoantibodieswerepreparedbyimmunizingthehealthymalerabbitswiththeintracellularproteinandstrainofPvindiflava,andthedetectionconditionofindirectELISAwereoptimized.Result:Thetiter
5、swererespectively1:32000and1:4000ofthetwopurifiedantibodiesfromstrain(J4)andtheintracellularprotein(P2).Theoptimumdilutionofthesecondaryantibodywasl:3000.ItwasshowedthatworkingconcentrationofJ4anditsantigenoptimumcoatingconcentrationwere1:16000and10。cfu/ml,andthatofP2werel:2000and10cfu/mlbyth
6、echecker—boardtests.Thetwoantibodiessensitivitywereboth10cfu/mL.whichhadhighspecificandnocrossreactionwithPseudomonassyringaepv.1aehrymansandtwenty—sixstrainssoon.Conclusion:ThespecificantibodiesofP.viriaiflavawereobtainedsuccessfullyinthisexperimentandaindirectELISAmethodforspecificdetection
7、ofP.viridiflavftwasestab-lished.Keywords:Pseudomonasviridiflava;antibodypreparation;titer;ELISA;detection黄瓜细菌性白枯病是由绿黄假单胞菌[Pseudomonasvirid-1.1.1.1菌种伽口(Burkholder1930)Dowson1939]引起的,是近年来辽宁黄瓜细菌性白枯病菌(Pseudomonasviridiflava),由作者实省黄瓜生产上发现的一种新的细菌病害
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