本文關(guān)鍵詞： 志賀氏菌 多克隆抗體 ELISA　出處：《華中農(nóng)業(yè)大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 志賀氏菌屬細(xì)菌屬于腸道致病菌,可引起臨床上嚴(yán)重的腹瀉。目前志賀氏菌的檢測方法有:常規(guī)檢測方法、分子生物學(xué)檢測方法、毒素檢測以及免疫學(xué)檢測方法等。常規(guī)檢測方法雖然準(zhǔn)確性好,但所需時(shí)間長;分子生物學(xué)方法快速、靈敏、特異性強(qiáng),且適用于不常見或新的病原微生物的檢測,但成本較高,且要求較高的技術(shù)水平;VITEK(全自動(dòng)細(xì)菌生化分析儀)、VIDAS(全自動(dòng)免疫分析儀)等雖然快速準(zhǔn)確、檢測量大,但儀器費(fèi)用頗高;免疫學(xué)中ELISA方法,具有操作簡便、快速、靈敏的優(yōu)點(diǎn)而廣泛應(yīng)用于致病菌的檢測。 本文通過對(duì)熱滅活的抗原免疫新西蘭大白兔,制備的抗血清,通過間接ELISA方法測得抗體效價(jià),發(fā)現(xiàn)免疫家兔50d后抗體逐漸上升,最終抗體效價(jià)為1:51200,采用辛酸-硫酸銨方法純化抗體得到純度較高的IgG。 通過對(duì)間接ELISA反應(yīng)條件的系列摸索,確定了各組分的最適工作條件。試驗(yàn)結(jié)果表明,Costar公司生產(chǎn)的酶標(biāo)板的變異系數(shù)為9.3%,達(dá)到ELISA的要求;最適封閉條件為5%脫脂牛乳,37℃,1h;抗體最適濃度為1.25μg/mL;抗體和HRP羊抗兔IgG的反應(yīng)時(shí)間均為37℃,1h;間接ELISA方法底物在室溫顯色5min,志賀氏菌檢測靈敏度為10~5～10~6cfu/mL。 通過對(duì)Dot-ELISA系列反應(yīng)條件的摸索,確定了各組分的最適工作條件。試驗(yàn)結(jié)果表明:最適封閉條件為5%脫脂牛乳,37℃,1h;抗體最適濃度為2.5μg/mL;抗體和HRP羊抗兔IgG的反應(yīng)時(shí)間均為37℃,1h;底物在室溫顯色15min,Dot-ELISA檢測志賀氏菌檢測靈敏度為10~6cfu/mL。 阻斷試驗(yàn)和交叉試驗(yàn)表明抗體不與沙門氏菌、變形桿菌、單增李斯特菌、金黃葡萄球菌、大腸桿菌、芽孢桿菌反應(yīng),具有良好的特異性。本研究建立的檢測方法,為志賀氏菌的檢測提供了行之有效的技術(shù)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，

本文編號(hào)：1475829