【作者】 杨旭东；

【导师】 师东方；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli, ETEC)是引起幼畜腹泻的主要病原,其主要致病因子有菌毛和肠毒素,不同动物源ETEC菌株可能带有F4(K88)、F5(K99)、F6(987P)或F41等菌毛。因此,对菌毛的检测不仅可以对幼畜腹泻进行诊断,同时也为选择特异性治疗方法提供科学的实验依据。带有F5菌毛的ETEC是引起仔猪黄痢和仔猪白痢的主要病原,建立一种针对F5菌毛的特异、简便、准确的检测方法对仔猪大肠杆菌病的诊断和防治具有重要意义。胶体金免疫层析技术的发展和应用,为建立F5菌毛检测方法提供了新的思路和技术基础。采用辛酸-饱和硫酸铵法和亲和层析柱法联合纯化抗F5菌毛蛋白的单克隆抗体和多克隆抗体。纯化后的单抗浓度为1.745mg/ml,经SDS-PAGE电泳分析IgG重链和轻链区带明显,腹水抗体效价由1.6×105上升为6.4×105。纯化后的多抗浓度为2.056mg/ml,经SDS-PAGE电泳分析IgG重链和轻链区带明显,血清抗体效价由3.2×104上升为6.4×104。纯化后的多抗和单抗可分别用于检测和标记。采用柠檬酸三钠法制备胶体金,通过检测证明金颗粒分布均匀,大小均一,适于标记抗体。用所制的金颗粒标记单克隆抗体,确定了抗体结合胶体金最佳结合量为54μg/ml,最适pH值为7.2。将金标抗体喷涂在结合垫上；在试纸条连接的硝酸纤维素膜上固定有抗F5菌毛蛋白的多克隆抗体作为检测线,羊抗鼠IgG的抗体作为质控线,组装试纸条。本研究对F5菌毛胶体金免疫层析试纸条的各项指标进行了优化,选择了效果较好的金标抗体稀释液(0.01M pH 7.2 PB(含0.5%BSA,1%海藻糖)、结合垫处理液(5%海藻糖含0.2%吐温-20)、封闭液(0.01M pH 7.2 Tris-cl含2% BSA,0.2%吐温-20)、样品垫处理液(0.02 MpH7.2 Tris-HCl含0.1%吐温-20)；硝酸纤维素膜(Sartorius CN 140)、结合垫(SB 06)、样品垫(SB 06)和吸收垫(S×27)；并确定了NC膜上检测线和质控线喷膜速度都为1.2μg/ml。经测试证明,本研究制备的F5菌毛胶体金免疫层析试纸条,最低检出量达到38.2μg/ml,试纸条与大肠杆菌的其它类型菌毛如：F4、F6、F41、F18ab无交叉反应,4℃条件下密封保存120天后仍能有效检出样品。用胶体金免疫层析试纸条对14份临床样品进行检测,结果与间接夹心ELISA、平板凝集实验、PCR方法所得结果符合率为100%。上述结果证明本研究制备的试纸条具有良好的敏感性、特异性和稳定性,可用于临床样品的检验。更多还原

【Abstract】 Diarrhea of piglets is frequently caused by porcine enterotoxigenic Escherichia coli (ETEC) including two types of virulence factors:fimbrial adhesins and enterotoxins. The adhesins of ETEC discovered from different animals maybe have F4 (K88), F5 (K99), F6 (987P), F41 and so on.Therefore, detection of fimbriae on animal yields can not only to diagnose the diarrhea, but also to provide the scientific experimental basis on treatment methods.F5+ETEC is the main pathogen which caused piglet yellow dysentery and white dysentery disease.It is important to establish a sensitive, convenient assay to detect F5 fimbriae. With the development and application of the immune colloidal gold chromatography, this assay provides new ideas and technology base for the establishment of F5 fimbriae detection.Polyclonal antibodies of F5 fimbriae and monoclonal antibody were purified by caprylic acid ammonium sulphate method and affinity chromatography. The concentration of purified monoclonal antibody and polyclonal antibody are 1.745mg/ml and 2.056mg/ml respectively. Through the polyacrylamide gel electrophoresis (SDS-PAGE) analyzing, they all have H line and L line obviously. And the potency of monoclonal antibody increase from 1.6×105 to 6.4×105,while the blood serum is from 3.2×104 to 6.4×104. Purified polyclonal antibody and monoclonal antibody can be used for detecting and tagging respectively.Colloidal gold which was prepared through deoxidization of trisodium citrate was uniformity under the electron microscope scanning so that they are sutiable to be used for tagging.The best combination of antibodies with colloidal gole is 54μg/ml, the optimum pH value is 7.2.Anti-F5 fimbriae monoclonal antibody labeled with colloidal gold was used as a detector.Anti-F5 fimbriae polyclonal antibody and sheep anti-mouse IgG were blotted on a nitrocellulose membrane for test line and control line, respectively.The various factors and conditions of the immunochromatographic lateral-flow assay were explored, and the optimal reaction conditions of assay were ascertained. Dilution of antibody tagged by gold particle(0.01M pH 7.2 PB,0.5% BSA,1% Trehalose)、Optimal investing solution of polyclonal antibody(0.01M, pH 7.2 Tris-cl)、confining liquid(0.01M pH 7.2 Tris-cl,2% BSA, 0.2%tween-20)、nitrocellulose filter(Sartorius CN140), Combine pad (SB 06),samplepad(SB 06),and absorption pad(S X 27)were chosen. And to determine the NC membrane test line and control line sprayed speed 1.2μg/ml.It was confirmed by experiment that the colloidal gold immunochromatographic strip of F5 fimbriae had good sensitivity (no response to F4、F6、F41、F18ab) and stability. The strip conserved at 4℃for 120 days could still detect the sample. The strip could be handled easily by untrained person and special instrument, and test result could be obtained in 5-10min, ideally suited for primary level and clinical diagnosis.更多还原