基于重组外膜蛋白P2的副猪嗜血杆菌抗体间接ELISA方法的建立  

Development of an Indirect ELISA Based on the Recombinant outer Membrane Protein (Omp) P2 for Detecting Antibodies against Haemophilus parasuis

作　　者：李淼[1,2] 李春玲[1,2] 叶严锋[1,2,3] 宋帅[1,2] 杨冬霞[1,2] 

机构地区：[1]广东省农业科学院兽医研究所,广东广州510640 [2]广东省兽医公共卫生公共实验室,广东广州510640 [3]华南农业大学兽医学院,广东广州510642

出　　处：《华北农学报》2011年第4期61-66,共6页Acta Agriculturae Boreali-Sinica

基　　金：广东省农业科技重大专项(2009A020101006);广州市农业科技重大专项(2009A1-E041)

摘　　要：旨在建立一种基于重组副猪嗜血杆菌(HPS)外膜蛋白(Omp)P2的检测副猪嗜血杆菌抗体的间接ELISA方法。将重组表达的Omp P2纯化后作为ELISA包被抗原,建立了一种基于重组P2蛋白的间接ELISA方法,并对此方法的反应条件进行了优化。确定ELISA的最佳条件为:抗原包被浓度为1.5μg/mL,被检血清1∶80稀释,辣根过氧化物酶标记的羊抗猪IgG(二抗)1∶4 000稀释。确定的阴性血清临界D450 nm值为0.231,阳性血清临界D450 nm值为0.280。将该ELISA方法与加拿大Biovet公司生产的副猪嗜血杆菌抗体检测试剂盒做相关比较,结果符合率为88.9%。本试验建立的方法具有良好的特异性,可以用于HPS抗体的检测。This experiment was conducted to detect the antibody of Haemophilus parasuis（HPS）based on the recombinant outer membrane protein（Omp） P2 through indirect ELISA.An indirect ELISA was developed and optimized with the purified recombinant HPS Omp P2 as coating antigen.The optimal conditions of ELISA were determined as follows.The microplate was coated with 150 ng of the recombinant P2 protein per well,and then blocked overnight at 4℃.The serum samples were diluted at 1∶ 80 and goat anti-pig HRP-IgG was diluted at 1∶ 4 000.The cutoff values at 450 nm of the negative or the positive sera were 0.231 or 0.280.The coincidence rate between the ELISA and Biovet HPS antibody test kit was determined as 88.9%.The results indicated that the indirect ELISA was specific and could be used for detecting anti-HPS antibodies.

关 键 词：副猪嗜血杆菌 重组OmpP2 间接ELISA 

分 类 号：S852.16[农业科学—基础兽医学]