副猪嗜血杆菌抗体间接ELISA检测方法的建立与应用.docVIP

目 录 摘 要 1 Abstract 2 前 言 3 1 材料与方法 4 1.1 菌种、血清及其他试剂 4 1.2 抗原的制备 4 1.3 间接ELISA的操作步骤 5 1.4 间接ELISA反应条件的确定 5 1.5 间接ELISA的特异性 6 1.6 间接ELISA的重复性 6 1.7 间接ELISA的临床应用 6 2.1 间接ELISA反应条件的筛选 6 2.2 间接ELISA的特异性 7 2.3 间接ELISA的重复性 7 2.4 间接ELISA的临床应用 7 3 讨论 7 参考文献 8 致 谢 9 附 录 10 副猪嗜血杆菌抗体间接ELISA检测方法的 建立与应用 摘 要 采用超声裂解的副猪嗜血杆菌菌体上清作为包被抗原，建立了检测副猪嗜血杆菌抗体的间接ELISA方法。通过对试验条件进行筛选优化，确定了最佳反应条件：抗原包被浓度80 ug/mL，37℃ 作用2 h后4℃过夜包被，待检血清的稀释度为1：3201 h，酶标二抗稀释度为l：15 0001 h，底物显色时间为15 min。待检血清S/N值≥2.5时判为阳性，S/N值2.5时判为阴性。特异性、重复性和敏感性试验以及对200份送检血清的检测结果表明，建立的间接ELISA方法的特异性和重复性良好，敏感性比间接血凝试验高，可用于副猪嗜血杆菌的检疫和流行病学调查。 关键词：副猪嗜血杆菌；抗体；间接酶联免疫吸附试验 Establishment and application of an indirect ELISA for detection of antibody against Haemophilus parasuis Abstract An indirect EL1SA coated with supernatant of sonicated Haemophilus parasuis serovar 5cell culture was established.The results of chessboard titration tests showed that coating antigen at a coneentratio of 80 ug/mL incubated optimally at 37℃ for 2 h and then coated overnight at 4℃.The optimaldilution of serum and HRP—IgG was 1 :320 and 1:1 5000,respectively.The reaction time of coating antigen with serum and serum with HRP—IgG was 1 h.The time of color development reaction was 1 5 min.Thecriteria evaluation was as follows:the serum was positive if S/N≥ 2.5,and the reverse is negative.Reproducibility,specificity,sensitivity tests and detection for 200 sera by the established ELISA confirmed thatthe developed ELISA had good stability and specificity.Compared with indirect haemagglutination test,the indirect ELISA was more sensitive.So this assay can be used as a tool for diagnosis and quarantine of H.parasuis. Keywords: Haemophilus parasuis；antibody；indirect ELISA (Haemophilus parasuis，HPS)Glasser’S病的病原体。Glasser’S病是一种以纤维素性浆膜炎、多发性关节炎、胸膜炎和脑膜炎为特征的猪呼吸道传染病，严重危害各年龄段的猪群，病死率较高[1]。近年来，该病日趋流行，危害日渐严重，已成为全球范围内影响养猪业的典型细菌性疾病。快速、准确地做出诊断是预防和控制该病的重要手段，本研究以超声裂解的HPS血清5型分离株菌液上清作为包被抗原，建立了一种快速、特异和灵敏的HPS抗体间接ELISA检测方法。 实验部分 1 材料与方法 1.1 菌种、血清及其他试剂 HPS血清5型分离株H3031菌种由本实验室保存。HPS 4型、5型阳性血清和阴