福建农业学报2016，Vol.31Issue(11)：1145-1150,6.DOI:10.19303/j.issn.1008-0384.2016.11.002
鳗弧菌单克隆抗体-胶体金检测方法的建立
Rapid Detection of Monoclonal Antibody against Vibrio anguillarum by Using Conjugated Colloidal Gold
摘要
Abstract
Vibrio anguillarum ,strain SMW5,were inactivated by formaldehyde to be used as the antigen for immunizing Balb/c mice. Three hybridomas strains secreting monoclonal antibodies (McAb ) against V . anguillarum were obtained and named as 1 H9,1C12,and 6F8 with the isotypes of IgG2a,IgM,and IgG2b, respectively.The titers of 1 H9 in ascites of Balb/c mice were 1∶6.4×10 5 .The polyclonal antibody was prepared against V .anguillarum,with a titer of 1 ∶ 3.2 × 10 5 .A rapid,accurate and easy gold immunochromatographic lateral flow assay methodology (GICA)was developed for detecting V .anguillarum.The monoclonal antibody, 1 H9,was conjugated with colloidal gold as a signal generator on the conjugate pad.The polyclonal antibody was used as a capture antibody for the test line (T),and the goat anti-mouse IgG antibody as the capture antibody for the control line (C)on nitrocellose membrane.A GICA test strip was assembled with an absorbing pad,a conjugate pad,and a nitrocellose membrane sprayed with the capture antibody.The sensitivity of the GICA test strip towards V .anguillarum was high with a detection limit of 6.3 × 10 4 cfu· mL-1 .The specificity of the assay showed no cross-reaction with 7 other aquaculture pathogens, including Aeromonas hydrophila ,A.cavia,A. sobria, Edwardsiella tarda ,and Streptococcus agalactiae .An accurate confirmation could be completed in 10 min for the assay.It appeared that the newly developed method using the GICA test strip was adequate for V .anguillarumdetection.关键词
鳗弧菌/单克隆抗体/多克隆抗体/胶体金/免疫层析Key words
Vibrio anguillarum/monoclonal antibody/polyclonal antibody/colloidal gold/gold immunochromatography assay分类
农业科技引用本文复制引用
吴斌,樊海平,王雪妹,黄小红,张新艳,叶小军,林志铿..鳗弧菌单克隆抗体-胶体金检测方法的建立[J].福建农业学报,2016,31(11):1145-1150,6.基金项目
现代农业产业技术体系建设专项(CARS-46-35) （CARS-46-35）
福建省产业技术开发项目[闽发改高技(2008-796)] （2008-796）
