Abstract:

Two methods including indirect ELISA and competitive ELISA were developed to rapidly detect a pathogen of Edwardsiella ictaluri in the early stage. A monoclonal antibody 5D11 was used as the detection antibody. In indirect ELISA when antigen was coaed at 37℃ overnight, the plate had good adsorption effect on bacteria. The optimal dilution of mAb and AP-goat-anti-mouse IgG were 1∶80 and 1∶1 000, respectively. The lowest concentration of E.ictaluri that could be detected was 5×106 cells/mL. Cross-reactivity test proved that the method had strong specificity. In competitive ELISA, the optimum working combination of coating antigen-mAb was 5×108~1∶80. The proportion of mAb and competitive antigen was 3∶7. A standard curve with R2=0.990 1 was established. This method had strong specificity, and the detection limit was 106 cells/mL. This method can qualitatively and quantitatively detect E.ictaluri within limits, which makes up the deficiency of indirect ELISA.

Key words: Edwardsiella ictaluri, detection, indirect ELISA, competitive ELISA

摘要：

为了对水产动物致病菌鮰爱德华菌进行早期、快速地检测,以鮰爱德华菌单克隆抗体5D11作为检测抗体,分别建立了间接ELISA和竞争ELISA两种快速检测鮰爱德华菌的方法,并用棋盘滴定法进行了条件的优化。结果显示,间接ELISA实验中采用37℃过夜这种包被方法能有效促进酶标板对细菌的吸附作用,单克隆抗体和二抗的最佳稀释倍数分别为1∶80和1∶1 000,病原菌的检测灵敏度为5×106 cells/mL,交叉反应实验证明该方法特异性强,并且可用于对鮰爱德华菌标准菌株及其分离株的快速检测;同时采用棋盘滴定法确定了竞争ELISA实验中包被抗原-抗体最佳工作组合为5×108~1∶80、抗体和竞争抗原比例为3∶7,并制作出了相关系数为0.990 1的标准曲线,该方法特异性强,最低检出限106 cells/mL,可在一定范围内对鮰爱德华菌进行定性和定量检测,弥补了间接ELISA的不足。

关键词: 鮰爱德华菌;检测;间接ELISA;竞争ELISA