桔青霉素单克隆抗体制备及三种真菌毒素蛋白芯片检测方法的研究

发布时间：2018-01-14 12:32

  本文关键词：桔青霉素单克隆抗体制备及三种真菌毒素蛋白芯片检测方法的研究　出处：《河北大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： 真菌毒素 单克隆抗体 蛋白芯片 检测方法

【摘要】：背景:真菌毒素为真菌产生的次级代谢产物,具有致癌、致突变、致畸等多种毒性,分布在小麦、玉米、饲料及多种食品中,一旦大量进入生态环境,将对动物、人及整个生物环境产生严重危害,因此倍受全球广泛关注。目前真菌毒素的检测方法主要有理化方法和免疫检测方法,理化方法灵敏度较高,但操作复杂,仪器昂贵,免疫方法一次只能检测一种毒素。因此,高通量的蛋白芯片方法将成为食品中真菌毒素快速检测的发展方向。 方法:本实验先通过抗原制备、抗原鉴定、免疫、细胞融合与筛选等步骤制备桔青霉素单克隆抗体,然后以桔青霉素(citrinin,CIT)、脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)和伏马菌素B1(fumonisin,B1)为研究对象,采用间接竞争免疫原理,以荧光信号为检测信号,确定CIT、DON和FB1人工抗原的最佳固定浓度、固定方式,抗CIT、抗DON、抗FB1三种单抗和IgG-Cy5二抗的最佳工作浓度,建立单独检测一种毒素的蛋白质芯片检测方法。并在此基础上初步建立同时检测三种真菌毒素的蛋白质芯片方法。 结果:(1)经过三种方法的鉴定,桔青霉素人工抗原偶联成功;(2)通过免疫小鼠制备杂交瘤细胞株,获得了一株抗桔青霉素单克隆抗体的细胞株;(3)建立了桔青霉素间接竞争ELISA,其IC50为96.2 ng/ml,检测限为10 ng/ml,线性范围为10-810 ng/ml;(4)对大麦、玉米、饲料样品进行添加回收实验,回收率分别为99-127.1%、75.4-124.5%、78-108%。(5)确定了CIT、DON和FB1人工抗原固定最佳工作浓度,分别为1:100、1:500、1:250;三种真菌毒素单克隆抗体的最佳工作浓度为1:500、1:400、1:4000;IgG-Cy5二抗的最佳工作浓度为1:1000,最佳人工抗原固定条件是25℃固定16 h,绘制了三种真菌毒素竞争抑制曲线,IC50分别为63.7 ng/mL、83.5 ng/mL、54.6 ng/mL,线性范围分别为:10-810 ng/mL、10-810 ng/mL、1.9-125 ng/mL。(6)绘制了三种真菌毒素同时检测的竞争抑制曲线,CIT灵敏度(IC50)为25.7 ng/mL,线性范围10-810 ng/mL,DON灵敏度(IC50)为58.5 ng/mL,线性范围10-810 ng/mL, FB1灵敏度(IC50)为24.9 ng/mL,线性范围1.9-125 ng/mL。 结论:通过制备抗桔青霉素单克隆抗体建立间接竞争ELISA,经添加回收试验初步验证该ELISA检测方法可用于实际样品的检测;根据我国规定的限量标准,初步建立的同时检测三种真菌毒素的蛋白芯片检测方法满足我国限量需求。
[Abstract]:Background: mycotoxins are secondary metabolites produced by fungi. They are carcinogenic, mutagenic and teratogenic. They are distributed in wheat, corn, feed and many kinds of food. It will cause serious harm to animals, human beings and the whole biological environment, so it has attracted worldwide attention. At present, the detection methods of mycotoxins are mainly physicochemical methods and immunoassay methods, and the sensitivity of physicochemical methods is high. But the operation is complicated, the instrument is expensive, the immune method can only detect one toxin at a time. Therefore, the high-throughput protein chip method will become the development direction of rapid detection of mycotoxins in food. Methods: the monoclonal antibody against citrinin was prepared by antigen preparation, antigen identification, immunity, cell fusion and screening, and then citrinin cit was used. Deoxynivalenolol (DON) and fumonisinB1 (fumonisinine B1) were used in the study. Using fluorescence signal as detection signal, the best fixed concentration, fixation mode, anti-CITand and anti-#en1# of CITDON and FB1 artificial antigens were determined. The optimum working concentration of three kinds of monoclonal antibodies and IgG-Cy5 second antibodies against FB1. A protein chip method for the detection of a single toxin was established and a protein chip method for the simultaneous detection of three mycotoxins was established. Results 1) after three methods of identification, the conjugation of citrinin artificial antigen was successful. (2) the hybridoma cell line was prepared by immunizing mice, and a cell line with monoclonal antibody against citrinin was obtained. (3) the indirect competition of citrinin was established, the IC50 was 96. 2 ng / ml, the detection limit was 10 ng / ml, and the linear range was 10-810 ng / ml; The recovery rates of barley, corn and feed samples were 99-127.1 and 75.4-124.5and 78-108.The recovery rate was 75.4-124.5. the CIT was determined. The optimal working concentration of DON and FB1 artificial antigen fixation was 1: 100 1: 500 and 1: 250, respectively. The optimal working concentration of monoclonal antibodies against three mycotoxins was 1: 500, 1: 400, 1: 4 000; The optimal working concentration of the second IgG-Cy5 antibody was 1: 1000, and the best condition of artificial antigen fixation was 25 鈩，

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