兔支气管败血波氏杆菌双夹心ELISA检测方法的建立  

Establishment of Double Sandwich ELISA for Detection of Bordetella bronchiseptica

作　　者：熊富强[1,2] 王芳[1] 恽时锋[2] 林焱[3] 范志宇[1] 胡波[1] 赵宁[2] 

机构地区：[1]江苏省农业科学院兽医研究所农业部动物疫病免疫与诊断重点开放实验室国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京军区南京总医院比较医学科,江苏南京210002 [3]南京农业大学动物医学院,江苏南京210095

出　　处：《江苏农业学报》2010年第1期120-125,共6页Jiangsu Journal of Agricultural Sciences

基　　金：农业部公益性行业科研专项(nyhyzx07-040);农业部现代农业产业技术体系建设专项(nycytx-44);江苏省科技支撑计划--农业部分(BE2008372);江苏省农业科技自主创新基金项目[CX(08)107]

摘　　要：为建立特异性好、敏感性高、稳定可靠的兔支气管败血波氏杆菌(Bb)双夹心ELISA检测方法,制备了兔抗Bb免疫血清和小鼠抗Bb腹水单抗(Bb McAb),并将它们提纯。用方阵法筛选最佳反应浓度,用特异性试验、敏感性试验和重复性试验对该方法进行鉴定,并与PCR检测方法比较。结果表明,兔抗Bb IgG的最佳稀释度为1∶6 400,浓度为2.5 mg/L;单抗的最佳稀释度为1∶800,浓度为5.0 mg/L。特异性试验和重复性试验结果表明,该方法特异性强、重复性好,与兔常见病菌大肠埃希氏菌、多杀性巴氏杆菌和产气荚膜梭菌均不发生反应,同时与PCR检测方法的符合率为100%。To establish a stable double sandwich ELISA method with good specificity and sensitivity for detection of Bordetella bronchiseptica （Bb）, rabbit anti-Bb hyperimmune serum and monoclonal antibody against Bb （Bb McAb）were prepared and purified. Chessboard titration crossing test was used to determine the optimal working concentrations of these two antibodies. Specificity, sensitivity and repeatability of the constructed ELISA were determined, and compared with PCR method further. The results showed that the best working concentration of the purified serum （2. 5 mg/L） and Bb MeAb （5.0 mz/L） were 1：6 400 and 1：800 ,respectively. The ELISA method developed in this study possessed high specificity and repeatability, with no cross-reaction with E. coli, P. psicicida and C. pelfringens. The coincidence of ELISA and PCR method was 100%.

关 键 词：支气管败血波氏杆菌 免疫血清 单克隆抗体 双夹心ELISA 

分 类 号：S858.291[农业科学—临床兽医学]