鲶爱德华氏菌间接酶联免疫快速检测法的建立

Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for Rapid Detection of Edwardsiella ictaluri

用鲶爱德华氏菌兔抗血清作为一抗,碱性磷酸酶(AP)标记的羊抗兔IgG作为酶标二抗,建立黄颡鱼"红头病"病原菌—鲶爱德华氏菌的间接酶联免疫(ELISA)快速检测法,并优化检测条件。抗原最佳包被浓度为107/mL,一抗工作的最佳稀释度为1∶211,病原菌的检测灵敏度为105/mL,交叉反应实验证明该方法特异性强,与迟钝爱德华氏菌、弧菌等13种标准菌株无交叉。应用上述技术对人工感染发病鱼中分离的优势菌进行检测,结果表明阳性检出率为80%;对自然发病黄颡鱼体内分离获得的20株优势菌检测结果表明,12株菌为鲶爱德华氏菌。

Indirect ELISA for detection of Edwardsiella ictaluri,pathogen of red-head disease of Pelteobagrus fulvidraco was established by using polyclonal antibody against E.ictaluri as primary antibody and alkaline phosphatase conjugated goat-anti-rabbit IgG as second antibody.The optimum coated concentration of the antigen was determined to be 107 cells/mL and the optimum polyclonal antibody concentration was determined to be 1∶211.The lowest concentration of E.ictaluri that can be detected was 105 cells/mL.Cross-reactivity test proved that the method was specificity,and had no cross-reaction with Edwardsiella tarda,Vibrio and other standard strains.With the established method to detect the superior strains isolated from artificial infected fish,the results showed that the positive rate was 80%.12 strains of 20 superior strains isolated from spontaneous diseased P.fulvidraco were identified as E.ictaluri.