【作者】 李鹏；

【导师】 赖卫华；

【作者基本信息】 南昌大学 ， 农产品加工与贮藏工程， 2007， 硕士

【摘要】 盐酸克仑特罗（Clenbuterol，CLB），俗称瘦肉精，是一种β₂-肾上腺素受体激动剂。作为治疗哮喘药物已经有30多年了。不幸的是这类药物被非法用来作为家畜的生长促进剂，用以改善营养物质在脂肪和肌肉之间的重新分配，增加瘦肉。这种非法应用会对食用残留有此类药物畜产品的消费者产生毒害作用，给人民的身体健康和生命安全造成潜在的危害。因此，世界上许多国家都禁止在畜牧业上使用盐酸克仑特罗。为了防止对人类的危害，必须建立快速、灵敏、有效的检测方法。ELISA方法检测盐酸克仑特罗残留是一种快捷、可靠的方法，可在实际检测中应用。噬菌体肽库展示技术近年来发展很快，可以用来淘选CLB替代抗原的模拟表位。这也为其它小分子半抗原物质的ELISA方法的建立提供新的思路。本论文主要包含两部分内容：1建立盐酸克仑特罗间接竞争ELISA检测方法。2用噬菌体肽库展示技术淘选盐酸克仑特罗替代抗原的模拟表位。第一部分包括盐酸克仑特罗偶联抗原的合成，单克隆抗体的纯化和效价的测定，ELISA各个影响因素最佳条件的优化，该ELISA方法性能的评价等。根据实验建立了盐酸克仑特罗间接竞争ELISA检测方法，包被偶联抗原为0.5ng／mL，加入CLB单抗原液稀释为1∶250000，酶标二抗原液稀释为1∶5000。其间接竞争标准曲线其线性范围为0.38—50ng／mL，IC₅₀为6.25ng／mL，检测下限为0.19ng／mL。曲线方程为y=35.149x+19.145，R=0.99378。批内和批间的变异系数分别为8.3％和9.97％。进行样品加标回收实验，平均回收率为93.14％。第二部分包括以抗CLB的单克隆抗体为靶分子，从噬菌体随机7肽库中进行4轮亲和淘选。从第4轮噬菌平板上随机挑选18个噬菌斑，经间接ELISA和间接竞争ELISA鉴定，挑选出6个合适的噬菌体。经测序可知其序全列为N—His—His—Thr—Phe—Phe—Lys—His—C。用噬菌体P8替代CLB抗原建立了间接竞争ELISA标准曲线，并用该噬菌体进行样品加标回收实验。更多还原

【Abstract】 Clbuterol （CLB） is one of theβ—Adrenergic agonist. It has beenused as bronchiodilators in human medicine for over 30 years.Unfortunately, it has been illegally used toenhance the growth, ratesof livestock and to promote the repartition from fat into muscle. Itmay lead to toxic effects after consumption of meat products containingCLB. A lot of countries in the world have banned the use of CLB so arapid and reliable method to detect CLB is needed. ELISA, which is arapid and sensitive detection method compared to other ones and can beused to screen CLB. Phage display is a new technology and has a rapiddevelopment. Itcan be applied in panning the Mimotopes of CLB as thesurrogate of CLB. This use of peptide libraries as sources of reagentsin immunoassays could be applicable in ELISA tests for other low-weighttoxic chemicals.The main results are as follows:1 Development of the indirect competitive ELISA of CLB.2 Panning mimotopes of CLB by phage display peptide library.Part one includes conjugating of CLB—BSA, detection of monoclonalantibody titers and purity, selection of ELISA factors, and evaluationof ELISA, etc. The method of ELISA was established by optimizing.parameters. The experiment results showed that the optimumconcentrations of BSA-CLB, monoclonal antibody against CLB andanti-mouse IgG were 0.5ng/mL, 1:250000 and 1:5000 respectively. Theindirect competitive ELISA curves were established, linear range of theinhibition curves was between 0.38ng/mL and 50ng/mL, IC₅₀ was 6.25 ng/mL,and the detectioh limit was 0.19ng/mL. The variation coefficients ofintra-assay and inter-assay were 8.3％and 9,97％respectively. Averagerecovery of CLB samples was 93.14％.In part two, the anti-CLB monoclonal antibody was used as ligand to pan the binding peptide from the Ph. D.-7^TM phage display peptide library.After four rounds of panning, 18 Clones were randomly picked from thefourth round. Those phage plaques were amplified and purified. Six of.the eighteen clones were identified positive by indirect ELISA. Thesequences of all. clones were His—His—Thr—Phe—Phe—Lys—His; Theindirect competitive ELISA curves were established for clone phage PS.Finally, ELISA was eastablished byclone phage which substitutes forCLB-BSA. Recovery of CLB samples was tested.更多还原