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

以灭活鳗弧菌(SMW5)全菌为抗原,应用杂交瘤技术,制备了鳗弧菌单克隆抗体3株,分别命名为1H9、1C12、6F8,细胞亚类分别为IgG2a、IgM、IgG2b,其中1H9腹水效价为1∶6.4×105。制备鳗弧菌(SMW5)兔多克隆抗体,效价为1∶3.2×105。应用纳米胶体金颗粒标记单克隆抗体1H9并制备金标垫,抗鳗弧菌(SMW5)兔多克隆抗体与羊抗鼠抗体作为捕获抗体包被硝酸纤维素膜,分别作为检测线T和质控线C,建立鳗弧菌的胶体金免疫层析快速检测方法。经过对试纸条的特异性和灵敏度测定,结果表明:试纸条与嗜水气单胞菌、温和气单胞菌、豚鼠气单胞菌、创伤弧菌、副溶血弧菌、副溶藻弧菌、迟缓爱德华氏菌,7种水产常见病原菌没有交叉反应,与鳗弧菌特异性反应,检测灵敏度为6.3×104cfu·mL^(-1),检测所需时间低于10min。所制备的鳗弧菌胶体金免疫层析检测试纸条具有快速、简便、特异性高和适应基层生产推广应用等优点。

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 1H9,1C12,and 6F8 with the isotypes of IgG2 a,IgM,and IgG2 b,respectively.The titers of 1H9 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,1H9,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 6.3×10^4cfu·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.