饲料中玉米赤霉烯酮的高效富集及免疫层析定量检测方法的建立

发布时间：2018-05-06 01:37

本文选题：胶体金免疫层析试纸条 + 玉米赤霉烯酮　；参考：《南昌大学》2015年硕士论文

【摘要】：玉米赤霉烯酮(zearalenone,ZEN)是由镰刀菌所产生的具有类雌激素作用的次级代谢产物。ZEN对食品和饲料的污染范围较为广泛,动物和人体摄入后,对机体具有一定的毒害作用,对畜牧养殖业和人体健康会造成很大的损害。因此,加强对食品及饲料中ZEN的监控检测显得尤为重要。由于饲料样本存在一定的基质干扰,对定量检测的结果会造成一定的影响,本文采用了聚乙烯亚胺-戊二醛介导体系制备了ZEN免疫磁性微球,建立了高效富集捕获饲料样本中ZEN的方法,以降低样本的基质效应对检测结果的影响。再以胶体金作为标记材料,研制出了胶体金免疫层析试纸条,结合HG-8胶体金试纸条读取仪,实现了对饲料中ZEN残留的快速定量检测。通过DCC/NHS活泼酯法制备了ZEN-BSA人工全抗原,并通过TLC监控和紫外全波段扫描验证ZEN-BSA成功合成,ZEN-BSA的浓度为2.55 mg/m L;用辛酸-硫酸铵法纯化了抗ZEN单抗,对抗体的性能进行了评价,测得效价为1:160000,浓度为5.36 mg/mL,通过SDS-PAGE电泳对纯化后的单抗进行了纯度分析,结果表明该单抗具有较高的纯度。为了保证捕获效率,本文以聚乙烯亚胺-戊二醛介导的方法制备了免疫磁性微球,对抗ZEN单克隆抗体的偶联量进行了优化,最佳偶联条件为:1 mg磁球表面偶联抗体100μg;对免疫磁富集捕获缓冲液的pH、甲醇浓度、捕获时间、微球加入量、洗脱液种类等条件进行了优化,最佳条件如下:将200μg PEI免疫磁球加入1 mL 20%甲醇PBS捕获缓冲液(pH7.0)中,在剧烈振荡条件下提取10 min,磁分离后用100μL 3%氨化甲醇洗脱磁球上的ZEN,洗脱液稀释复溶后用ELISA试剂盒检测。通过样本加标回收试验的结果表明,当ZEN加标量为50-250μg/kg时回收率逐渐上升,且在250μg/kg时,回收率达到最高,为93.28%。本文以胶体金作为标记物,优化了胶体金的粒径、抗体标记的pH值、抗体的标记量、T线全抗原的包被浓度、C线二抗的包被浓度和金标抗体的喷量等几个参数,确定了定量检测ZEN胶体金免疫层析试纸条的最佳生产工艺条件:胶体金粒径为40 nm,标记pH为6.0,抗体标记量为4μg/mL,结合垫上的金标抗体喷量为2μL/cm,NC膜上T线ZEN-BSA的包被浓度为0.8 mg/mL,C线上驴抗鼠二抗的包被浓度为0.4 mg/mL。由此,成功地研制出ZEN胶体金免疫层析定量检测试纸条,定量检测时间为15 min,该试纸条具有良好的特异性和稳定性,最低灵敏度为2.559μg/L,IC50=9.09μg/L。加标回收率在67.70%—104.48%之间。此外,采用ELISA试剂盒评估了本法的准确性,两种方法的相关性较好。
[Abstract]:Zen, a secondary metabolite produced by Fusarium oxysporum, has a wide range of contamination of food and feed. After ingested by animals and human beings, Zen has a certain toxic effect on the body. It will do great harm to animal husbandry and human health. Therefore, it is particularly important to strengthen the monitoring and detection of ZEN in food and feed. ZEN immunomagnetic microspheres were prepared by polyethyleneimide-glutaraldehyde (Glutaraldehyde) mediated system because of some matrix interference in feed samples. In order to reduce the effect of matrix effect on the detection results, a method of high efficiency enrichment and capture of ZEN in feed samples was established. Using colloidal gold as labeling material, a colloidal gold immunochromatographic strip was developed. Combined with HG-8 colloidal gold strip reader, the rapid quantitative determination of ZEN residues in feed was realized. The artificial antigens of ZEN-BSA were prepared by DCC/NHS active ester method, and the concentration of ZEN-BSA was 2.55 mg/m L by TLC monitoring and UV scanning. The anti-ZEN monoclonal antibody was purified by octanoic acid-ammonium sulfate method, and the performance of the antibody was evaluated. The titer was 1: 1600 and the concentration was 5.36 mg / mL. The purity of the purified McAb was analyzed by SDS-PAGE electrophoresis. The results showed that the McAb had high purity. In order to ensure the capture efficiency, the immunomagnetic microspheres were prepared by polyethyleneimide-glutaraldehyde mediated method, and the coupling amount of monoclonal antibody against ZEN was optimized. The optimal coupling condition was 100 渭 g for the surface coupling antibody of magnetic sphere at 1 mg, the pH of the magnetic enrichment capture buffer, the concentration of methanol, the capture time, the amount of microspheres and the type of eluent were optimized. The optimum conditions were as follows: 200 渭 g PEI immunomagnetic sphere was added to 1 mL 20% methanol PBS capture buffer (pH 7.0), extracted under the condition of intense oscillation for 10 min, then separated by magnetic separation and eluted by 100 渭 L 3% amination methanol on the magnetic sphere. The eluent was diluted and redissolved and detected by ELISA kit. The results of the standard recovery test showed that the recovery rate increased gradually when the scalar concentration of ZEN was 50-250 渭 g/kg, and the recovery rate reached the highest level at #number0# 渭 g/kg, 93.28 渭 mol 路mol ~ (-1) 路L ~ (-1) 路min ~ (-1) 路min ~ (-1). In this paper, the colloidal gold was used as a marker to optimize the particle size of colloidal gold, the pH value of antibody labeling, the concentration of T line whole antigen coated with antibody and the coating concentration of second antibody of C line and the amount of spray of gold labeled antibody. The optimum production conditions for quantitative detection of ZEN colloidal gold immunochromatographic strip were determined as follows: colloidal gold particle size was 40 nm, labeling pH was 6.0, antibody labeling amount was 4 渭 g / mL, and the amount of gold-labeled antibody sprayed on the pad was 2 渭 L / cm ~ (-1) of T line ZEN-BSA coated on NC membrane. The coating concentration of the donkeys anti mouse second antibody on the line of 0.8 mg / mL C was 0.4 mg / mL. Therefore, the ZEN colloidal gold immunochromatographic quantitative test strip was successfully developed. The quantitative detection time was 15 min. The test strip had good specificity and stability, and the lowest sensitivity was 2.559 渭 g / L IC50 / 9 渭 g / L, and the minimum sensitivity was 2.559 渭 g / L ~ (50) and 9.09 渭 g 路L ~ (-1) 路L ~ (-1). The recoveries were between 67.70% and 104.48%. In addition, the ELISA kit was used to evaluate the accuracy of the two methods.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816;O652

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