黄海希瓦氏菌单抗介导间接ELISA快速检测技术的建立

Establishment of monoclonal antibody mediated indirect ELISA for detection of Shewanella smarisflavi

应用农业部海洋水产增养殖学重点实验室制备的特异性抗黄海希瓦氏菌Shewanella smarisflavi AP629单克隆抗体3D9作为一抗,以碱性磷酸酶(AP)标记的羊抗鼠Ig作为酶标二抗,建立了仿刺参Apostichopusjaponicus"化皮病"病原菌——黄海希瓦氏菌AP629的间接ELISA快速检测方法,并进行了条件优化。结果表明:抗原的最佳包被浓度为107个/mL,一抗工作的最佳稀释度为1∶32,病原菌的检测灵敏度为5×105个/mL。该方法特异性强,与希瓦氏菌属其它种类、弧菌和气单胞菌等均无交叉反应。该方法的建立有助于快速准确地诊断由黄海希瓦氏菌AP629引起的仿刺参疾病。

Indirect ELISA pathogen of ulcer skin in sea cucumber Apostichopus japonicus was established for detection of Shewanella smarisflavi（AP629） using monoclonal antibody 3D9（MAb 3D9） against strain AP629 as primary antibody and alkaline phosphatase conjugated goat-anti-mouse Ig as second antibody.The optimum coated concentration of the antigen was found to be 107 cells/mL,and the optimum MAb concentration 1∶32.The minimal detectable concentration limit of strain AP629 was 5×105 cells/mL.Examination of cross-reactions of MAb 3D9 with other bacteria showed that no cross reactions could be detected.With the developed indirect ELISA,homogenate of body wall and muscles from the diseased sea cucumber and isolated bacteria were detected,indicating that strain AP629 could be detected from body wall and muscle of diseased sea cucumber,and homogenate of muscle could be used directly in indirect ELISA.