志贺氏菌多克隆抗体制备及ELISA检测方法的建立

发布时间：2018-01-30 08:56

  本文关键词： 志贺氏菌 多克隆抗体 ELISA　出处：《华中农业大学》2008年硕士论文　论文类型：学位论文

【摘要】： 志贺氏菌属细菌属于肠道致病菌,可引起临床上严重的腹泻。目前志贺氏菌的检测方法有:常规检测方法、分子生物学检测方法、毒素检测以及免疫学检测方法等。常规检测方法虽然准确性好,但所需时间长;分子生物学方法快速、灵敏、特异性强,且适用于不常见或新的病原微生物的检测,但成本较高,且要求较高的技术水平;VITEK(全自动细菌生化分析仪)、VIDAS(全自动免疫分析仪)等虽然快速准确、检测量大,但仪器费用颇高;免疫学中ELISA方法,具有操作简便、快速、灵敏的优点而广泛应用于致病菌的检测。 本文通过对热灭活的抗原免疫新西兰大白兔,制备的抗血清,通过间接ELISA方法测得抗体效价,发现免疫家兔50d后抗体逐渐上升,最终抗体效价为1:51200,采用辛酸-硫酸铵方法纯化抗体得到纯度较高的IgG。 通过对间接ELISA反应条件的系列摸索,确定了各组分的最适工作条件。试验结果表明,Costar公司生产的酶标板的变异系数为9.3%,达到ELISA的要求;最适封闭条件为5%脱脂牛乳,37℃,1h;抗体最适浓度为1.25μg/mL;抗体和HRP羊抗兔IgG的反应时间均为37℃,1h;间接ELISA方法底物在室温显色5min,志贺氏菌检测灵敏度为10~5～10~6cfu/mL。 通过对Dot-ELISA系列反应条件的摸索,确定了各组分的最适工作条件。试验结果表明:最适封闭条件为5%脱脂牛乳,37℃,1h;抗体最适浓度为2.5μg/mL;抗体和HRP羊抗兔IgG的反应时间均为37℃,1h;底物在室温显色15min,Dot-ELISA检测志贺氏菌检测灵敏度为10~6cfu/mL。 阻断试验和交叉试验表明抗体不与沙门氏菌、变形杆菌、单增李斯特菌、金黄葡萄球菌、大肠杆菌、芽孢杆菌反应,具有良好的特异性。本研究建立的检测方法,为志贺氏菌的检测提供了行之有效的技术手段。
[Abstract]:Shigella is a kind of intestinal pathogenic bacteria, which can cause severe diarrhea in clinic. At present, the detection methods of Shigella are routine detection method and molecular biological detection method. Toxin detection and immunological detection methods. Although the accuracy of routine detection method is good, but the time required is long; Molecular biological methods are rapid, sensitive, specific and suitable for the detection of rare or new pathogenic microorganisms, but the cost is high and the technical level is high. VITEK (automatic Bacteriological and biochemical Analyzer), etc., is fast and accurate, but the cost of the instrument is quite high. ELISA method in immunology has been widely used in the detection of pathogenic bacteria because of its advantages of simplicity, rapidity and sensitivity. The antiserum of New Zealand white rabbits was immunized with heat-inactivated antigens. The titers of antibodies were determined by indirect ELISA method. It was found that the antibodies increased gradually after 50 days of immunization. The final titer of the antibody was 1: 51200.The antibody was purified by octanoic acid-ammonium sulfate method to obtain IgG with high purity. The optimum working conditions of each component were determined by exploring the series of indirect ELISA reaction conditions. The results showed that the coefficient of variation of the enzyme label plate produced by Costar was 9.3%. Meet the requirements of ELISA; The optimum sealing condition was 5% skim milk at 37 鈩，

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