【摘要】 试验从患典型烂鳃病草鱼病变部位分离到1株柱状黄杆菌(FC-3株),采用0.4%甲醛灭活制成免疫原,接种健康120日龄桃源蛋鸡,收集鸡蛋并提纯卵黄抗体(IgY),并以此IgY为基础建立间接ELISA方法。结果:用FC-3株制备的IgY经初步提纯后电泳分离可见重链和轻链2条蛋白条带,获得了IgY多克隆抗体。建立的间接ELISA方法检测柱状黄杆菌纯培养液的检出限值为1×10~7cfu/mL,抗原包被最佳浓度和抗体最佳工作浓度分别为1∶100和1∶512,辣根过氧化物酶标记兔抗鸡IgY最佳工作浓度为1∶4 000。结论:建立的间接ELISA方法对柱状黄杆菌有明显的检测特异性,与其他菌株无交叉反应,重复性好。更多还原

【Abstract】 Streak plate method was used to separate the pathogenic strain FC-3 from diseased regions of Ctenopharyngodon idella with gill rot,named Flavobacterium columnare. The strain was inactivated by 0.4% formaldehyde according to the immune plan to stimulate 120 days laying Taoyuan hens’ the production of Yolk antibody( IgY). The high level immuned eggs were collected. The purified IgY was used to establish indirect ELISA for detecting the FC-3 strain. Results: the Flavobacterium columnare( FC-3) IgY was preliminary purified,2 protein strips heavy chain and light chain were visible by Electrophoresis separation. This indicate the IgY polyclonal antibody was acquired. Titer of the polystyrene chloride microtitration plates were coated with Flavobacterium columnare antigen were detected by IgY indirect ELISA at the amount of 1×107 cfu/mL. The working concentration of antigen,primary antibody and HRP-labelled rabbit antichicken IgY were 1 ∶100,1 ∶512 and 1 ∶4 000. Conclusion: the detection specificity of the method was also determined,Flavobacterium columnare can be detected,while other bacteria strains can not be detected,good repeatability.更多还原