大肠杆菌O157-H7论文：快速检测大肠杆菌O157-H7胶体金免疫层析方法的建立

1、大肠杆菌O157:H7论文：快速检测大肠杆菌O157:H7胶体金免疫层析方法的建立【中文摘要】大肠杆菌O157：H7(Escherichia coli O157:H7)是引起出血性肠炎和溶血性尿路综合症的主要致病菌。牛肉、奶制品、蔬菜等食物是其主要传播途径,除此之外,动物和人、人和人的接触也可能导致大肠杆菌O157：H7感染。由于其感染发病快、死亡率高,严重影响了人民的身体健康。因此,快速诊断大肠杆菌O157：H7具有重要的现实意义。本文旨在建立快速、特异性强、适用于大量样品和现场检测的大肠杆菌O157：H7胶体金免疫层析方法。本文采用全菌抗原和细胞碎片抗原分别免疫日本大耳兔,制备了抗大肠杆菌

2、O157：H7多克隆抗体。获得的多克隆抗体效价达106。分别用辛酸-硫酸铵法和磁珠吸附法对多克隆抗体进行纯化,纯化后的抗体通过SDS-PAGE进行蛋白分析,并用斑点杂交对纯化后的抗体进行特异性分析。结果显示,辛酸-硫酸铵法纯化后的抗体仍有较多的杂蛋白,而磁珠吸附法能有效去除这些杂蛋白,得到较纯的抗体。纯化后的多克隆抗体均与其它一些细菌有交叉反应。本文采用胶体金标记商品化的单克隆抗体,通过把多克隆抗体和驴抗鼠抗体(二抗)喷涂于硝酸纤维素膜分别作为检测线(T线)和质控线(C线),研制了大肠杆菌O157：H7胶体金快速检测试纸条,并对其进行了评价。通过实验,确定了试纸条工艺条件如下：标记pH为8.0

3、、蛋白标记量为25g/mL、金标抗体离心转速为4000 r/min、胶体金封闭剂为PEG 20000、T线喷涂浓度为1mg/mL。C线喷涂浓度为1.5mg/mL。用纯培养细菌对试纸条的灵敏度和特异性进行了评价,结果显示试纸条的灵敏度为105个细胞/mL,试纸条除与金黄色葡萄球菌(CMCC26003)和鼠伤寒沙门氏菌(ATCC 13311)有弱阳性反应外,与其它24株常见的细菌无交叉反应。37加速保存实验结果表明,本试纸条常温保质期在15个月以上。在25 g牛肉或莴苣样品中添加10100 CFU的菌,37增菌6h后,试纸条检测为阳性结果。为了进一步提高试纸条的特异性和灵敏度,本文采用提取的大肠杆

4、菌O157：H7鞭毛蛋白免疫Balb/c小鼠,经细胞融合、筛选、克隆化共得到4株能稳定分泌抗大肠杆菌O157：H7单克隆抗体的杂交瘤细胞株,并制备了相应的腹水。配对结果显示,有5对抗体可用于试纸条的制备。【英文摘要】Escherichia coli (E. coli) O157:H7 is an important foodborne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome in human. Human infections with E. coli O157:H7 have usually

5、 been found to follow the consumption of contaminated, improperly prepared beef products, dairy foods, and vegetables. In addition, direct animal-to-person and person-to-person contact can result in E. coli O157:H7 transmission. It has seriously affected the health of human because its fast invasion

6、 and high mortality when infections. Hence, it has important practical significance of developing a rapid method for diagnosis E. coli O157:H7. In this study, a colloid gold immunochromatographic method which is rapid, high specificity and suitable for a great number of samples and on site detection

7、 was established for detection of E. coli O157:H7.Japanese white rabbits were immunized with the whole cell and cell debris of E. coli O157:H7 to prepare polyclonal antibody. The titer of antibody was 106. Caprylic acid ammonium sulphate precipitation and magnetic beads adsorption were used to purif

8、y polyclonal antibodies (pAb). Then SDS-PAGE was used for proteins analysis while Dot blot hybridization was used for analysis the specificity of pAb. The SDS-PAGE results showed that antibodies purified by caprylic acid ammonium sulfate precipitation still had many other proteins; however, the magn

9、etic beads adsorption could effectively remove the other proteins and get pure immunoglobulin. The antiserum after purified reacted with other bacteria.In this study, commercial anti-E. coli O157:H7 monoclonal antibody was labeled with colloidal gold and anti-E. coli O157:H7 polyclonal antibody and

10、donkey anti-mouse IgG were dispensed on the nitrocellulose membrane as the test line and control line respectively. The strips parameters were as follows:The reaction system pH was 8.0, anti-E. coli O157:H7 monoclonal antibody labeling with gold particles was 25g/mL, the gold-antibody conjugate was

11、centrifuged at 4000 rpm/min and blocked with PEG 20000, lmg/mL polyclonal antibody and 1.5 mg/mL donkey anti-mouse IgG were dispensed for the test line and control line respectively. The senitivity and specificity of the strip were determined using pure cultured bacteria. E. coli O157:H7 could be de

12、tected at a minimum of 105 cells/mL. Cross reaction test with 26 bacteria strains showed that only Staphylococcus aureus (CMCC 26003) and Salmonella typhimurium (ATCC 13311) had slight cross reaction, whereas the rest 24 strains had no cross reaction. The preservation test at 37indicated that the st

13、rips could be stored at room temperature for more than 15 months. The strips showed positive result after inoculating E. coli O157:H7 in samples (beef or lettuce) at 10 to 100 CFU/25 g and incubating for 6 h.In order to increase the specificity and sensitivity of the assay, Balb/c mice were immunize

14、d with flagellum proteins isolated from E. coli O157:H7. After fusion and cloning, four hybridoma cells which would stable secret antibodys against E. coli O157:H7 were abtained and prepared ascitic fluid. The matching results showed that 5 pairs of antibodies could be used for strip preparation.【关键

15、词】大肠杆菌O157:H7 多克隆抗体 胶体金 免疫层析 单克隆抗【英文关键词】Escherichia coli O157:H7 polyclonal antibody colloidal gold immunochromatography monoclonal antibody【目录】快速检测大肠杆菌O157:H7胶体金免疫层析方法的建立摘要3-4ABSTRACT4-5缩略语表6-11第一章 引言11-201.1 E. coli O157:H7的临床表现和感染11-121.2 E. coli O157:H7的特异性抗原和抗体制备研究进展12-141.2.1 E. coli O157:H7的O

16、抗原121.2.2 E. coli O157:H7的H抗原12-131.2.3 其它特异性抗原13-141.3 E. coli O157:H7检测方法的研究进展14-171.3.1 常规检测方法14-151.3.2 基于PCR的分子生物学方法15-161.3.3 免疫学检测方法16-171.3.4 新兴技术171.4 胶体金快速检测技术的概述17-191.4.1 胶体金的性质181.4.2 胶体金制备方法181.4.3 胶体金标记抗体18-191.4.4 免疫胶体技术在微生物检测中的应用191.5 展望19-20第二章 抗大肠杆菌O157:H7多克隆抗体的制备20-312.1 前言202.2

17、材料与方法20-252.2.1 菌株、免疫动物202.2.2 主要试剂和仪器设备202.2.3 相关试剂和培养基配置20-212.2.4 E. coli O157:H7抗原制备21-222.2.5 其它菌株包被原的制备222.2.6 动物免疫22-232.2.7 间接ELISA232.2.8 E. coli O157:H7多克隆抗体效价监测232.2.9 抗体纯化23-242.2.10 多克隆抗体特异性评价24-252.3 结果与讨论25-312.3.1 E. coli O157:H7全菌抗原和细胞碎片抗原25-262.3.2 多克隆抗体免疫应答曲线26-272.3.3 纯化多抗电泳分析27-

18、282.3.4 多克隆抗体特异性28-292.3.5 讨论29-31第三章 大肠杆菌O157:H7胶体金快速检测试纸条的制备31-423.1 前言313.2 材料与方法31-353.2.1 菌株313.2.2 主要试剂和仪器设备313.2.3 相关试剂与培养基配制31-323.2.4 多克隆抗体和单克隆抗体配对32-333.2.5 胶体金制备333.2.6 标记pH值的确定333.2.7 蛋白标记量的确定33-343.2.8 标记胶体金离心参数343.2.9 封闭剂的确定343.2.10 检测线包被浓度的确定34-353.2.11 质控线包被浓度的确定353.3 结果与讨论35-423.3.1

19、 多克隆抗体和单克隆抗体配对35-363.3.2 制备的胶体金分析36-373.3.3 标记pH值的确定37-383.3.4 蛋白标记量的确定383.3.5 标记胶体金离心参数38-393.3.6 封闭剂的确定393.3.7 检测线包被浓度的确定39-403.3.8 质控线包被浓度的确定40-413.3.9 讨论41-42第四章 大肠杆菌O157:H7胶体金快速检测试纸条的评价42-524.1 前言424.2 材料与方法42-454.2.1 菌株424.2.2 主要试剂和仪器设备424.2.3 相关试剂与培养基配制42-434.2.4 试纸条的制备434.2.5 细菌培养与处理434.2.6 阴性样品测试434.2.7 试纸条特异性43-444.2.8 试纸条灵敏度444.2.9 食品样品检测444.2.10 试纸条保质期44-454.3 结果与讨论45-524.3.1 试纸条阴性样品测试454.3.2 试纸条特异性45-474.3.3 试纸条灵敏度分析47-484.3.4 试纸条保质期48-494