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β-兴奋剂多残留快速检测技术的研究

标题: β-兴奋剂多残留快速检测技术的研究
英文标题: Study on the Method for Rapid Multi-determination of β-adrenergic Agonist
作者: 张亚峰
出版时间: 2009-01-01
所在大学: 河北农业大学
关键词: 沙丁胺醇,克伦特罗,多克隆抗体,ELISA,胶体金
英文关键词: Salbutamol,clenbuterol,Polyclonal antibody,ELISA,colloidal gold
论文级别: 硕士
学位: 学位论文
导师: 刘聚祥%王建平
专业: 基础兽医学
提交时间: 2009
摘要: β2-兴奋剂(β2-adrenergic agonist, BAA)属于拟肾上腺素类药物,由于其具有促进动物生长、提高瘦肉率的作用曾被作为饲料添加剂应用于动物养殖业中.然而BAA长期作为饲料添加剂后,它潜在的危害就逐渐被人们认识,会严重危害消费者的身体健康,因此,建立BAA残留检测的方法成为关系食品安全的亟待解决的问题. 本文分别采用混合酸酐法和碳化二亚胺法合成了沙丁胺醇(SAL)免疫抗原和包被抗原,SAL与牛血清白蛋白(BSA)的偶联比分别为12:1和6:1,与卵清蛋白(OVA)的偶联比分别为9:1和4:1.用免疫抗原免疫兔子后,所得抗血清经饱和硫酸铵法沉淀和DEAE-纤维素阴离子交换层析纯化,得到纯度较高的抗SAL抗体IgG.该抗体对SAL有较高的特异性,IC50为6.004 ng/mL,同时对沙丁胺醇的同系物克伦特罗(CL)有85.44%的交叉反应率,IC50为7.027 ng/mL,但对其他β-兴奋剂类药物无交叉反应性. 以该抗体为基础建立了同时检测沙丁胺醇和克伦特罗的间接竞争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4万倍和8万倍稀释.以抑制率为纵坐标,分别以SAL和CL浓度的负对数为横坐标绘制标准曲线,针对SAL和CL的线性方程分别为Y=-20.868X+96.359和Y=-20.77X+94.722.该ELISA方法对SAL和CL的最低检测限分别为0.219ng/mL和0.253ng/mL.在空白猪肉和猪尿中按1-50ng/g(mL)的浓度添加SAL和CL后,对SAL的添加回收率为72.2%-87.8%,变异系数分别为4.6%-11.7%,对CL的回收率为79.9%-97.4%,变异系数为1.3%-13.5%. 以该沙丁胺醇抗体为基础,结合免疫胶体金技术,制备了快速检测沙丁胺醇和克伦特罗的金标试纸条.试纸条直接浸入猪尿中即可在5min内用肉眼对结果进行判定,阳性结果为一条红线,阴性结果为两条红线.试纸条对猪尿中沙丁胺醇和克伦特罗的最小检出限分别为2.0ng/mL和2.5ng/mL. 本研究以沙丁胺醇抗体为基础建立了一种快速,灵敏的ELISA方法,并研制了一种简便、快速的沙丁胺醇免疫胶体金检测试纸条,以期能更加快速有效的监控养殖业中沙丁胺醇和克伦特罗的非法使用,保障动物源性食品安全.
英文摘要: β_2 -adrenergic agonists have the effect of enhancing animal growth and increasing protein synthesis, which are used as feed additives in animal breeding. However, the residues ofβ_2 -adrenergic agonists in animal tissues would be harmful to consumer, the residue detection was a important work assotiation of food safety.In this study, salbutamol (SAL) was coupled to bovine serum albumin (BSA) and ovalbumin (OVA) by use of mixed anhydride method and the carbodiimide method to prepare the immunogen and coating antigen, respectively. The coupling ratio of SAL with BSA was 12:1 and 6:1, respectively, and those of with OVA was 9:1 and 4:1, respectively. The immunogen was used to immunize the rabbits, and the anti-SAL antibody were obtained and purified by the saturated ammonium sulfate precipitation method and the DEAE-cellulose anion-exchange chromatography purification. The anti-SAL antibody has high specificity to salbutamol with IC50 of 6.004 ng/mL, and showed a crossreactivity of 85.44%toward clenbuterol with IC50 of 7.027 ng/mL, but no crossreactivity toward otherβ-agonists.Based on the antibody, an indirect competitive ELISA method for detection of salbutamol and clenbuterol was developped. Optimum concentrations of coating antigen and antibody were determined by board titration as 40,000 and 80,000-fold dilution. The standard inhibition curves for SAL and CL were developped use inhibition rate as longitudinal coordinates and negative logarithm of concentrations as abscissa with the linear equations of Y=-20.868X+96.359 for SAL and Y=-20.77X+94.722 for CL. The limit of detection of the ELISA method for SAL and CL was 0.219 ng/mL and 0.253 ng/mL, respectively.After fortification of SAL and CL in the blank swine muscle or urine at levels of 1-50ng/g(mL), the samples were determined by the ELISA. Recoveries for SAL were in the range of 72.2%-87.8%with coefficients of variation of 4.6%-11.7%, and recoveries for CL were in the range of 79.9%-97.4%with coefficients of variation of 1.3%-13.5%.Furthermore, a colloidal gold chromatographic strip for rapidly detection of SALand CL based on the anti-SAL antibody was prepared. For detection, the strip was directly dipped into the urine and the result was observed within 5 min, and the positive result was determined as the appearance of one red line and the negative result was two red line. The limit of detection for SAL and CL were 2.0ng/mL and 2.5ng/mL in swine urine, respectively.In this paper, a simple, rapid and sensitive ELISA method and a colloidal gold strip based on anti-SAL polyclonal antibody for simultaneous detection of SAL and CL were developped. These methods could be used to quickly and efficiently monitor the illegal use of SAL and CL in animal breeding with the aim of protection the quality of animal-derived food.