抗副溶血弧菌抗体的制备及免疫学检测方法的研究

发布时间：2018-05-28 09:43

本文选题：副溶血弧菌 + 多克隆抗体　；参考：《华中农业大学》2010年硕士论文

【摘要】：副溶血弧菌属于肠道致病菌,可以引起强烈腹泻或者致死等症状。目前,检测副溶血弧菌的方法有：传统检测方法、免疫学检测方法、分子生物学检测方法和仪器检测方法。传统检测方法准确,但需要的时间较长；分子生物学检测方法快速、灵敏、特异性强,但需要较高的成本和较高的专业技术；仪器检测方法快速准确,但需要的成本高；免疫学检测方法具有操作简便、快速、灵敏等优点而广泛应用于致病菌的检测。本研究利用副溶血弧菌菌体抗原作为免疫抗原,利用免疫学技术制备抗副溶血弧菌的多克隆抗体和单克隆抗体,对抗副溶血弧菌多克隆抗体间接ELISA检测方法的条件进行了优化；同时通过对胶体金和金标抗体的研究,成功制备副溶血弧菌免疫层析试纸条,对副溶血弧菌进行快速检测。研究内容有： 1.抗副溶血弧菌多克隆抗体的制备与纯化选取副溶血弧菌菌株ATCC17802作为免疫抗原,经沸水浴2 h后制备菌体抗原,免疫新西兰大白兔,得到抗副溶血弧菌多克隆抗体。经过辛酸硫酸铵沉淀盐析法纯化后的抗体效价为1：25600,抗体中蛋白质含量为8 mg/mL, SDS-PAGE蛋白质分子量为156 kD。 2.抗副溶血弧菌多克隆抗体间接ELISA条件的优化通过对间接ELISA条件的优化得出各组分最佳的工作条件。结果为：抗原的最佳包被条件为碳酸盐缓冲液4℃包被过夜,浓度为108cfu/mL,最佳封闭条件为5%脱脂奶粉37℃封闭0.5 h,一抗的最佳稀释度为1：3200,37℃反应1 h,酶标二抗的最佳稀释度为1：2000,37℃反应1 h,底物最佳反应时间是10 min。在此条件下,抗副溶血弧菌多克隆抗体仅与副溶血弧菌出现阳性反应,与鲍氏志贺氏菌、甲型副伤寒沙门氏菌、奇异变形杆菌、单增李斯特菌、金黄葡萄球菌、大肠杆菌、枯草芽孢杆菌、产气肠杆菌、铜绿假单胞菌、白念珠菌、肺炎克雷伯菌创伤弧菌、梅氏弧菌、溶藻弧菌和霍乱弧菌均呈阴性反应。间接ELISA对副溶血弧菌最低检出量为107cfu/mL。 3.抗副溶血弧菌单克隆抗体的制备与纯化以相同的免疫抗原免疫Bal b/C小鼠,制备抗副溶血弧菌单克隆抗体。制备的小鼠腹水经过辛酸提取法纯化,间接ELISA的效价为1：400,SDS-PAGE蛋白质分子量约为156 kD。 4.胶体金和金标抗体的制备以柠檬酸三钠还原法制备胶体金,得到分布良好的40 nm的胶体金溶液。通过二次差速离心法,成功把抗副溶血弧菌多克隆抗体与胶体金偶联。 5.副溶血弧菌免疫层析试纸条的制备及研究利用抗副溶血弧菌的多克隆抗体和单克隆抗体,应用胶体金免疫层析技术制备副溶血弧菌免疫层析检测试纸条。通过对试纸条材料玻璃纤维素膜和硝酸纤维素膜的研究,得到制备检测试纸条的最佳材料为玻璃纤维素膜Ahlsorm 8694和硝酸纤维素膜Sartorius CN 140.检测试纸条控制线和检测线的包被抗体分别为1 mg/mL羊抗兔IgG和1 mg/mL抗副溶血弧菌单克隆抗体。检测试纸条分别通过与弧菌属内5种菌、属外10菌以及种内不同食品分离到的分离株进行反应,证实具有较好的特异性。人工模拟实验证实检测试纸条在食品检测过程中具有较好的实用性。检测试纸条的检测限为106cfu/mL。室温避光干燥保藏3个月,仍然具有良好的稳定性。
[Abstract]:Vibrio parahaemolyticus is a kind of intestinal pathogenic bacteria, which can cause severe diarrhea or fatal symptoms. At present, the methods of detecting Vibrio parahaemolyticus are traditional detection methods, immunological detection methods, molecular biological detection methods and instrument detection methods. Traditional detection methods are accurate but need longer time; the method of molecular biological detection is fast. It is fast, sensitive and specific, but requires high cost and high professional technology. The method of instrument detection is fast and accurate, but the cost is high. The immunoassay method has the advantages of simple operation, rapid and sensitive, and it is widely used in detection of pathogenic bacteria.
In this study, the polyclonal and monoclonal antibodies against Vibrio parahaemolyticus were prepared using the antigen of Vibrio parahaemolyticus as immune antigen, and the conditions of the indirect ELISA detection method against Vibrio parahaemolyticus were optimized. At the same time, the parahaemolyticus was successfully prepared by the study of colloidal gold and gold antibody. Vibrio immunochromatographic strip was used for rapid detection of Vibrio parahaemolyticus.
Preparation and purification of polyclonal antibody against Vibrio parahaemolyticus 1.
The strain ATCC17802 of Vibrio parahaemolyticus was selected as the immune antigen, and the bacterial antigen was prepared after 2 h boiling water bath. The polyclonal antibody of Vibrio parahaemolyticus was obtained by immunizing New Zealand white rabbits. The antibody titer was 1:25600 after the acid ammonium sulfate precipitation method was purified. The protein content of the antibody was 8 mg/mL, and the molecular weight of SDS-PAGE protein was 156. KD.
Optimization of indirect ELISA conditions for 2. polyclonal antibodies against Vibrio parahaemolyticus
The optimum conditions of each component were obtained by optimizing the indirect ELISA conditions. The result was that the optimum envelope of the antigen was 4 centigrade, the concentration was 108cfu/mL, the best closed condition was 5% closed 0.5 h, the best dilution was 1 h at 1: 3200,37, and the best dilution of the enzyme standard two. The reaction time was 1 h at 1:2000,37 C, and the best reaction time of the substrate was 10 min., and the polyclonal antibody of Vibrio parahaemolyticus was only positive with Vibrio parahaemolyticus, and it was gas producing with Bao Shizhi, Salmonella paratyphi A, Proteus mirabilis, M. monocytogenes, Staphylococcus aureus, Escherichia coli, Bacillus subtilis. Enterobacteriaceae, Pseudomonas aeruginosa, Candida albicans, Klebsiella pneumoniae, Vibrio merivibrio, Vibrio alginolyticus and Vibrio cholerae were negative. The minimum detectable amount of indirect ELISA to Vibrio parahaemolyticus was 107cfu/mL.
Preparation and purification of monoclonal antibodies against Vibrio parahaemolyticus 3.
The monoclonal antibody against Vibrio parahaemolyticus was prepared by immunizing Bal b/C mice with the same immune antigen. The mouse ascites was purified by octanic acid extraction. The titer of indirect ELISA was 1:400, and the molecular weight of SDS-PAGE protein was about 156 kD..
Preparation of 4. colloid gold and gold antibody
Colloidal gold was prepared by reduction of sodium citrate three. A colloidal gold solution with a good distribution of 40 nm was obtained. By two differential centrifugation, polyclonal antibodies against Vibrio parahaemolyticus were successfully combined with colloidal gold pairs.
Preparation and study of 5. immunochromatographic strip for Vibrio parahaemolyticus
Using the polyclonal and monoclonal antibodies against Vibrio parahaemolyticus, the immunochromatography test paper of Vibrio parahaemolyticus was prepared by colloidal gold immunochromatography. Through the study of the glass cellulose membrane and the nitrocellulose membrane of the test paper material, the best material for the preparation of the test paper was Ahlsorm 8694 and nitrate of the glass cellulose membrane. The acid cellulose membrane Sartorius CN 140. detected the monoclonal antibodies against the control lines and the detection lines of the test strips and the detection lines were 1 mg/mL Sheep anti rabbit IgG and 1 mg/mL anti Vibrio parahaemolyticus, respectively. It is proved that the test paper strip has good practicability in the process of food testing. The detection limit of test paper is 3 months at room temperature and still has good stability at room temperature for 3 months.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【相似文献】