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饲料和动物性食品中三聚氰胺检测技术研究

标题: 饲料和动物性食品中三聚氰胺检测技术研究
英文标题: Study on the Methods for Detection of Melamine in Animal Feeds and Animal Derived Foods
作者: 钟永本
出版时间: 2011-01-01
所在大学: 河北农业大学
关键词: 三聚氰胺,环丙胺嗪,ELISA,免疫亲和色谱,HPLC
英文关键词: melamine,cyromazine,ELISA,immuno affinity chromatography,HPLC
论文级别: 硕士
学位: 学位论文
导师: 刘聚祥%王建平
专业: 基础兽医
提交时间: 2011
摘要: 本研究合成了三聚氰胺半抗原,并采用混合酸酐法合成了免疫抗原和包被抗原.半抗原与牛血清白蛋白、卵清蛋白的偶联比分别为10:1和8:1.用免疫抗原免疫新西兰白兔,所得血清经饱和硫酸铵-辛酸沉淀法纯化抗体IgG.以该抗体为基础建立了一种间接竞争ELISA方法.方阵滴定法确定包被抗原和抗体的最适工作浓度分别为4000和1000倍稀释.该方法对三聚氰胺有较高的特异性,同时对环丙胺嗪有59.7%的交叉反应率.以抑制率为纵坐标,分别以三聚氰胺和环丙胺嗪浓度的对数为横坐标绘制标准曲线,针对三聚氰胺和环丙胺嗪的线性方程分别为Y=-24.938X+106.53,Y=-23.856X+109.46.该ELISA方法对三聚氰胺和环丙胺嗪的IC50分别为185 ng/mL和310 ng/mL,对三聚氰胺和环丙胺嗪的最低检测限分别为4.6ng/mL和6.5ng/mL.在空白饲料、牛奶中按1-125ng/g(mL)的浓度添加三聚氰胺,添加回收率分别为84.5%-98.03%和61.6%-78.72%,变异系数分别为2.0%-2.9%和4.2%-9.6%.在鸡肉、鸡蛋中按1-125ng/g(mL)的浓度添加环丙胺嗪,添加回收率分别为80.44-97.39%和75.64-93.25%,变异系数分别为1.0%-4.9%和1.4%-3.3%.以该抗体为基础制备了免疫亲和色谱柱(IAC),确定了IAC柱的上样、洗涤、洗脱及再生条件.该柱对三聚氰胺的动态柱容量为1250ng,绝对柱容量为125ng,且每根IAC柱可连续使用30次后,柱容量仍可达320ng.然后,以IAC柱为样品前处理技术建立了三聚氰胺残留检测的IAC-HPLC法,对三聚氰胺的检测限为15ng/mL,定量限为25ng/mL.以15,30,105,150ng/g的浓度在空白牛奶、鸡肉、鸡蛋中添加三聚氰胺,回收率分别为90.7%-101.4%、86.0%-97.3%、84.7%-93.6%,变异系数分别为11.1%-16.5%、8.8%-17.9%、11.9%-19.7%.通过比较,证明IAC柱的净化效果优于常规固相萃取柱的净化效果.因此,本研究建立的ELISA法可以作为快速筛选方法,IAC-HPLC法作为确证性方法用于动物性食品中三聚氰胺残留的日常监控.
英文摘要: In this study, the hapten of melamine was synthesized and the hapten was then coupled to bovine serum albumin and ovalbumin by use of mixed anhydride method to prepare the immunogen and coating antigen. The coupling ratio of melamine to bovine serum albuin was 10:1, and that of to ovalbumin was 8:1. The immunogen was used to immunize the rabbits and the obtained serum was purified by the saturated ammonium sulfate-octylic acid precipitation method.Then, an indirect competitive ELISA method was developped for simultaneous detection of melamine and cyromazine. Optimum concentrations of coating antigen and antibody were determined by board titration as 40,000 and 1,000-fold dilution. The antibody showed high specificity to melamine, and showed a crossreactivity of 59.7%toward cyromazine. The standard inhibition curves for melamine and cyromazine were developped by using of inhibition rate as longitudinal coordinates and logarithm of concentrations as abscissa. The linear equations for melamine and cyromazine were Y=-24.938X+106.53 and Y=-23.856X+109.46, respectively. The IC50 values for melamine and cyromazine were 185 ng/mL and 310 ng/mL, and the limit of detection for melamine and cyromazine was 4.6 ng/mL and 6.5 ng/mL, respectively.After fortification of melamine in the blank feed, milk, at levels of 1-125ng/g(mL), the samples were determined by the ELISA. Recoveries for melamine were in the range of 84.5%-98.03%and 61.6%-78.72%with coefficients of variation of 2.0%-2.9%and 4.2%-9.6%, after fortification of cyromazine in chicken, eggs at levels of 1-125ng/g(mL), the samples were determined by the ELISA. Tnd recoveries for cyromazine were in the range of 80.44-97.39%and 75.64-93.25%with coefficients of variation of 1.0%-4.9%and 1.4%-3.3%.An affinity chromatography column (IAC) was prepared based on the antibody and the conditions of loading, washing, eluting and regeneration were evaluated. The dynamic column capacity for melamine was 1250 ng per mL of gel and the absolute column capacity was 125 ng/mg IgG. The column could be used successively for 30 times with column capacity still up to 320 ng/mL per mL of gel. The IAC-HPLC procedure for determination of melamine was firstly developed with the limit of detection of 15ng/mL and the limit of quantification of 25 ng/mL, respectively. At fortified levels of 15, 30,105,and 150μg/kg, the recovery of melamine in bank blank feed, milk, chicken, eggs, were 90.7%-101.4%, 86.0%-97.3%, 84.7%-93.6%with coefficients of variation of 11.1%-16.5%, 8.8%-17.9%, 11.9%-19.7%. Furthermore, the purification performance of IAC was better than that of the conventional SPE.Therefore, the ELISA method present in this study could be used as rapid screening method and the IAC-HPLC could be used as confirmation method for the routine determination of melamine residues in animal derived products.