恩诺沙星、苏丹红及氯霉素残留的酶联免疫检测方法的建立
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摘要
近年来,社会经济发展迅猛,人们的生活水平得到了很大的提高,人们对各种食品的需求量也越来越大,因此,一些不法养殖户和商贩在食品中会非法添加一些兽药以提高产量或美化外观。恩诺沙星、苏丹红及氯霉素等由于价格低廉、添加后效果好,常被非法添加到食品中。这些药物添加后的残留可能有潜在的致癌性,致畸性或使人及动物产生耐药性,因此这些药物的残留得到了各国食品安全部门的重视。为了给消费者提供一个安全的食品环境,各国都建立了一些检测药物残留的方法,如：高效液相色谱法(HPLC),气相色谱-质谱联用法(GC-MS)等。但是这些仪器方法通常价格都很昂贵,并且样品处理复杂、耗时长,因此在日常快速检测中不易普及。酶联免疫吸附分析法(ELISA)由于其快速、灵敏度高等优点,受到各国分析者的青睐,应用前景广泛,在此基础上建立的磁分离方法(MAIA),结合了抗原抗体反应的专一性及磁分离的高效性,使得此方法具有更高的灵敏度及应用前景。本研究主要进行了恩诺沙星及苏丹红的酶联免疫分析及氯霉素的两种磁分离方法的比较。
恩诺沙星残留的检测方法已经很多了,本试验主要运用恩诺沙星的试剂盒检测市场上销售的多种动物源性食品,以确定商品的质量。经检测,恩诺沙星试剂盒的灵敏度(IC50)为3.237 ng.mL-1,检测限为0.42 ng.mL-1,工作曲线范围为0.5-13.5 ng.mL-1。样本的检测结果为：市售动物源性食品中,肝脏中恩诺沙星的残留量较大。
对于苏丹红,其抗原通过戊二醛的方法合成,免疫原用牛血清白蛋白(BSA)与半抗原偶联,包被抗原用卵清蛋白(OVA)与半抗原偶联,制备完成后,用制得的免疫原免疫Balb/c小鼠,经过一系列步骤制得单克隆抗体。再经过一系列的条件优化,建立间接竞争ELISA法评价抗体：灵敏度(IC5o)为1.7 ng.mL-1,工作曲线的线性范围为0.5-13.5 ng.mL-1,抗体的专一性较好,在辣椒油中的回收率为82%-91.8%,在辣椒酱中的回收率为59%-103.8%,批内变异系数在6.5-8.8%之间,批间变异系数为7.7%左右。本试验与其它的抗原合成方法相比,本方法更为简单,中间步骤少,产率较高,能很好的用于ELISA检测。
对于氯霉素,通过两种磁分离方法评价抗体,比较了这两种方法的优劣。用普通ELISA方法检测时,抗体的灵敏度(IC50)为0.72 ng.mL-1；用第一种磁分离方法(MethodⅠ)检测时,抗体的灵敏度为0.05 ng.mL-1,工作曲线范围为0.005-5.0 ng.mL-1；用第二种磁分离方法(MethodⅡ)检测时,抗体的灵敏度(IC50)为0.4 ng.mL-1,工作曲线的范围为0.01-5.0 ng.mL-1。两种方法检测牛奶中氯霉素的回收率为80-106%,变异系数为4.7-15%,两种方法相比,MethodI的灵敏度更高,线性范围更广,但是MethodII的操作更简单,更适于日常检测,可以开发成为一种通用试剂。
通过对这几种药物的分析研究,我们成功的制备了苏丹红的单克隆抗体,并建立了这几种药物的酶联免疫吸附分析方法及氯霉素的磁分离方法。本论文的研究新进展：用新方法制备了苏丹红的抗原和单克隆抗体,并建立了较稳定的ELISA检测方法；用恩诺沙星抗体检测了市售的动物源性食品中恩诺沙星的残留,明确了动物源性食品在恩诺沙星方面的安全情况；比较了两种磁分离方法的优劣,为试剂盒的开发奠定了基础。
With the development of the economy, the living standard is improving, and the requirement of the animal derived food is increasing. In order to increase the output of the animal derived food and make the food beautiful to look at, some veterinary drugs were added by lawless persons. But most of the drugs were banned to be used in food processing because of their carcinogenicity or other side effects. In order to ensure the safety of the foodstuff, many detection methods were developed to detect the drug residue, such as LC-MS, GC-MS and HPLC. Because of their fund and time consumption, these methods were hard to be used widespread. Enzyme-linked immunosorbent assay (ELISA) was a rapid detection method to detect drug residue with a high sensitivity and low detection limit. And magnetic affinity immunoassay (MAIA) was a method developed on the basement of the ELISA with a higher sensitivity. The perpurse of the thesis was to develop some ELISA methods to detect the enrofloxacin and sudan red in food and make sure which MAIA method was suitable to make kits.
Enrofloxacin was detected by the ELISA kits with an IC50 of 3.237 ng.mL"1 in buffer. And the detection limit was 0.42 ng.mL-1. The aim of this thesis was to detect the enrofloxacin residue in animal derived food. The experiment proved that the enrofloxacin residues were more in liver than in muscle, and the most of the animal derived food on sale were injected with enrofloxacin.
Sudan red was a kind of aze dyes which was banned to be used in food. The immunogen was synthesized by glutaraldehyde. And the monoclonal antibody was obtained by cell culture and monoclone. The antibody showed high sensitivity with an IC50 of 1.7 ng.mL-1, and the range of the working curve was from 0.5 ng.mL-1 to 13.5 ng.mL-1. The specificity of the antibody was evaluated by the cross-reactivity of the antibody with sudan red and para red. The detection limit in chilli oil and chilli jam was 9.0 ng.mL-1 and 19.6 ng.mL-1, respectively. The recovery rates were in the range of 80%-110%, and the variation were both less than 20%.
MAIA was based on ELISA with higher sensitivity. Compared with the ELISA (IC50=0.72 ng.mL"1), the sensitivity of the MAIA to chloramphenicol was 0.05 ng.mL-1 in MethodⅠand 0.4 ng.mL-1 in MethodⅡThe experiments showed that the sensitivity of MethodⅠwas higher than MethodⅡwhile MethodⅡwas suitable to be used in kits production because of its convenience.
In this thesis, we obtained monoclonal antibody of sudan red and developed the detection methods to the drugs. Based on this thesis, some ELISA kits can be produced.
恩诺沙星残留的检测方法已经很多了,本试验主要运用恩诺沙星的试剂盒检测市场上销售的多种动物源性食品,以确定商品的质量。经检测,恩诺沙星试剂盒的灵敏度(IC50)为3.237 ng.mL-1,检测限为0.42 ng.mL-1,工作曲线范围为0.5-13.5 ng.mL-1。样本的检测结果为：市售动物源性食品中,肝脏中恩诺沙星的残留量较大。
对于苏丹红,其抗原通过戊二醛的方法合成,免疫原用牛血清白蛋白(BSA)与半抗原偶联,包被抗原用卵清蛋白(OVA)与半抗原偶联,制备完成后,用制得的免疫原免疫Balb/c小鼠,经过一系列步骤制得单克隆抗体。再经过一系列的条件优化,建立间接竞争ELISA法评价抗体：灵敏度(IC5o)为1.7 ng.mL-1,工作曲线的线性范围为0.5-13.5 ng.mL-1,抗体的专一性较好,在辣椒油中的回收率为82%-91.8%,在辣椒酱中的回收率为59%-103.8%,批内变异系数在6.5-8.8%之间,批间变异系数为7.7%左右。本试验与其它的抗原合成方法相比,本方法更为简单,中间步骤少,产率较高,能很好的用于ELISA检测。
对于氯霉素,通过两种磁分离方法评价抗体,比较了这两种方法的优劣。用普通ELISA方法检测时,抗体的灵敏度(IC50)为0.72 ng.mL-1；用第一种磁分离方法(MethodⅠ)检测时,抗体的灵敏度为0.05 ng.mL-1,工作曲线范围为0.005-5.0 ng.mL-1；用第二种磁分离方法(MethodⅡ)检测时,抗体的灵敏度(IC50)为0.4 ng.mL-1,工作曲线的范围为0.01-5.0 ng.mL-1。两种方法检测牛奶中氯霉素的回收率为80-106%,变异系数为4.7-15%,两种方法相比,MethodI的灵敏度更高,线性范围更广,但是MethodII的操作更简单,更适于日常检测,可以开发成为一种通用试剂。
通过对这几种药物的分析研究,我们成功的制备了苏丹红的单克隆抗体,并建立了这几种药物的酶联免疫吸附分析方法及氯霉素的磁分离方法。本论文的研究新进展：用新方法制备了苏丹红的抗原和单克隆抗体,并建立了较稳定的ELISA检测方法；用恩诺沙星抗体检测了市售的动物源性食品中恩诺沙星的残留,明确了动物源性食品在恩诺沙星方面的安全情况；比较了两种磁分离方法的优劣,为试剂盒的开发奠定了基础。
With the development of the economy, the living standard is improving, and the requirement of the animal derived food is increasing. In order to increase the output of the animal derived food and make the food beautiful to look at, some veterinary drugs were added by lawless persons. But most of the drugs were banned to be used in food processing because of their carcinogenicity or other side effects. In order to ensure the safety of the foodstuff, many detection methods were developed to detect the drug residue, such as LC-MS, GC-MS and HPLC. Because of their fund and time consumption, these methods were hard to be used widespread. Enzyme-linked immunosorbent assay (ELISA) was a rapid detection method to detect drug residue with a high sensitivity and low detection limit. And magnetic affinity immunoassay (MAIA) was a method developed on the basement of the ELISA with a higher sensitivity. The perpurse of the thesis was to develop some ELISA methods to detect the enrofloxacin and sudan red in food and make sure which MAIA method was suitable to make kits.
Enrofloxacin was detected by the ELISA kits with an IC50 of 3.237 ng.mL"1 in buffer. And the detection limit was 0.42 ng.mL-1. The aim of this thesis was to detect the enrofloxacin residue in animal derived food. The experiment proved that the enrofloxacin residues were more in liver than in muscle, and the most of the animal derived food on sale were injected with enrofloxacin.
Sudan red was a kind of aze dyes which was banned to be used in food. The immunogen was synthesized by glutaraldehyde. And the monoclonal antibody was obtained by cell culture and monoclone. The antibody showed high sensitivity with an IC50 of 1.7 ng.mL-1, and the range of the working curve was from 0.5 ng.mL-1 to 13.5 ng.mL-1. The specificity of the antibody was evaluated by the cross-reactivity of the antibody with sudan red and para red. The detection limit in chilli oil and chilli jam was 9.0 ng.mL-1 and 19.6 ng.mL-1, respectively. The recovery rates were in the range of 80%-110%, and the variation were both less than 20%.
MAIA was based on ELISA with higher sensitivity. Compared with the ELISA (IC50=0.72 ng.mL"1), the sensitivity of the MAIA to chloramphenicol was 0.05 ng.mL-1 in MethodⅠand 0.4 ng.mL-1 in MethodⅡThe experiments showed that the sensitivity of MethodⅠwas higher than MethodⅡwhile MethodⅡwas suitable to be used in kits production because of its convenience.
In this thesis, we obtained monoclonal antibody of sudan red and developed the detection methods to the drugs. Based on this thesis, some ELISA kits can be produced.
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