摘要
以大菱鲆Scophthalmus maxinzoa出血性败血症病原菌迟钝爱德华氏菌Edwardsiella tarda为抗原，从免疫家兔体内获得高免疫血清，建立快速检测大菱鲆出血性败血症病原迟钝爱德华氏菌的间接ELISA技术。采用棋盘滴定法确定间接ELISA抗原和抗血清的最适工作浓度分别为10^7个／mL和1：20000；最低检测菌浓度为10^4个／mL；抗血清与其他细菌标准菌株的交叉反应结果均为阴性，表明该方法具有较高的特异性。将该方法优化后检测了19尾人工感染出血性败血症并发病的大菱鲆和19尾健康的大菱鲆，阳性检测率分别为89．5％和的10．5％。同时在间接ELISA方法的基础上建立竞争ELISA检测技术，结果表明：理想的包被抗原浓度为10^7个／mL，兔抗血清的工作浓度为1：20000，酶标抗体工作浓度为1：1000，可测最适范围为2×10^5～2×10^8个／mL，最低检测菌浓度为2×10^5个／mL，得到回归方程y=-0．3811x＋2．2335，R^2=0．992。
An indirect Enzyme- Linkd Immunosorbent Assay （ELISA） has been developed for rapid diagnosis of Edwardsiella tarda, a bacterial pathogen of hemorrhagic septicemia of turbot Scophthalmus maximus at the best working antigen concentration of 10^7 cell/m with antiserum of 1 ： 20 000 by checkerboard titration. The sensitivity of the serum was tested, and the lowest Edwardsiella tarda suspension was kept at a rate of 104 cell/mL. Cross negative reaction of antisera with other type strain bacteria and different inhibition showed that this optimized method detected the infected and normal turbot with positive detection rate of 89.5% and 10.5%, separately. The results showed that that the assay was used to detect not only the infected turbot, but also the carriers of Edwardsiella tarda. The optimal concentration of the coating antigen was 10^7 cell/mL. The dilutions of polyclonal antibody against Edwardsiella tarda and sheep anti - rabbit IgG were 1 ： 20 000 and 1 ： 1000 separately. Quantization of the Edwardsiella tarda was linear from 2 × 10^5 - 2 × 10^8 cell/mL with the detection limit of 2 × 10^5 celt/mL and expressed as the equation y = - 0. 3811x ＋ 2. 2335 （R^2 = 0.992） and there was a standard curve.
出处
《大连水产学院学报》
CSCD
北大核心
2008年第4期252-257,共6页
Journal of Dalian Fisheries University
基金
辽宁省教育厅高等学校科研计划项目(2004D239)
作者简介
王斌（1962-），女，教授。E-mail：Wangbin@dlfu．edu．cn
