莱克多巴胺、氯霉素、三聚氰胺药物残留的酶联免疫检测法的建立
摘要
随着食品贸易国际化的发展,人们对动物源性食品的需求量大大增加,因而畜牧养殖业得到了迅速发展。但是,由于科学知识的缺乏和经济利益的驱使,在养殖业中超标使用药物和滥用违禁药物的现象普遍存在,在我国情况尤为严重。近几年,我国食品出口因药物残留问题被许多国家退货或者销毁,给我国造成了巨大的经济损失,严重损害了我国食品的质量形象。药物残留不仅直接对人体产生急、慢性毒性作用,引起细菌耐药性的增加,还通过环境和食物链的作用间接对人体健康造成潜在威胁；药物残留对人体的影响主要表现在变态反应和过敏反应、致畸、致突变和致癌作用等方面。动物源性食品中违禁药物的残留问题也越来越成为全世界共同关注的公共卫生问题。
做好食品安全工作不仅是保障人类健康,促进畜牧业发展和我国对外贸易的需要,也是国家政治经济稳定的需要。控制动物源性食品中药物残留已经成为目前确保动物源性食品安全的一项重要措施,许多经济发达国家十分重视药物残留监控,颁布了一系列畜产品药物残留管理法规,制定了详尽的药物残留限量标准,严格实施对畜产品药物残留的监控、监督与检测。近年来,我国也高度重视这一问题,为了有效地控制动物源性产品中的药物残留,制定了严格的残留标准和休药期以限制违禁药物在食品中的残留,采取法律、经济、行政和技术手段实行监控,并借鉴国外先进经验和技术,结合我国国情,研究、筛选出一套可行的违禁药物在食品中的残留检测方法。
目前国内外采用的残留分析检测方法主要有理化检测法(包括HPLC、LC-MS、LC-MS/MS等仪器法)、微生物检测法、免疫检测法(包括ELISA、RIA、EIA、FIA、MAIA)等。理化检测法准确、稳定、可靠,可以作为标准方法,但仪器设备昂贵,样品前处理复杂,费时,费力,不易普及；微生物检测法往往会高出最高检测量(MRLs),灵敏度不高。因此必须建立快速、灵敏、有效的检测方法。免疫检测法中的酶联免疫检测法(包括ELISA、CL-ELISA、MAIA等)克服了以上方法的缺点,是一种快速、灵敏、方便、易普及的检测方法,有着广阔的发展前景。本论文旨在建立莱克多巴胺的化学发光酶联免疫检测法(CL-ELISA)、氯霉素的磁分离酶联免疫检测法(MATA)和三聚氰胺的酶联免疫检测法(ELISA)
在对莱克多巴胺的研究中,首先根据其结构特点,利用对氨基苯甲酸法合成了免疫原(Rac-cBSA),将此免疫原免疫新西兰大白兔制得莱克多巴胺的多克隆抗体,合成免疫原的同时,我们用BDE法合成了包被抗原(Rac-OVA)。然后利用间接CL-ELISA研究莱克多巴胺抗体的亲和度、灵敏度、交叉反应及在牛奶中莱克多巴胺回收率的测定。经检测,IC50是0.45 ppb,检测限为0.001 ppb,线性范围为0.01-10 ppb；该抗体对其它p-兴奋剂交叉反应率很小；莱克多巴胺在牛奶中的添加回收率在78-120%之间,批内变异系数低于18.1%,批间变异系数低于24.7%。
在对氯霉素的研究中,通过直接MAIA法测得氯霉素单克隆抗体的亲和度、灵敏度、交叉反应及在牛奶中氯霉素回收率的测定。经检测,氯霉素抗体的IC50是0.05ppb,线性范围为0.005-5 ppb；该抗体仅对琥珀氯霉素有交叉反应(交叉反应率为6.25%),对其它的酰胺醇类抗生素的交叉反应率很小；检测氯霉素在牛奶中的添加回收率在85.2-113.7%之间,变异系数在9.9-22%之间。
在对三聚氰胺的研究中,首先根据其结构特点,利用水溶性碳二亚胺法合成了免疫原(MEL-TG),将此免疫原免疫小鼠,经过细胞融合与克隆化得到三聚氰胺的单克隆抗体,合成免疫原的同时,我们用同样的方法合成了包被抗原(MEL-BSA)。然后利用间接ELISA法研究了三聚氰胺抗体的灵敏度、交叉反应及在牛奶中三聚氰胺回收率的测定。经检测,IC50是22.6 ppb,检测限为5.0 ppb,线性范围为5.0-405 ppb；该抗体对其它类似物交叉反应率很小；三聚氰胺在牛奶中的添加回收率在70-125%之间,批内变异系数低于12.0%,批间变异系数低于13.0%。
通过本课题的研究,成功制备了用于检测莱克多巴胺的多克隆抗体和检测三聚氰胺的单克隆抗体,利用这些抗体,本研究建立了莱克多巴胺的化学发光酶联免疫检测法、氯霉素的磁分离酶联免疫检测法和三聚氰胺的酶联免疫检测法,并且验证了化学发光酶联免疫法和磁分离酶联免疫检测法比起传统的比色酶联免疫检测法具有更好的灵敏度和检测下限,为最终开发商品化的国产化试剂盒奠定了良好的基础,并为其它药物抗体的制备和检测试剂盒的开发开辟了道路。
With the development of international trade in food, the need for the animal-origin food is increasing greatly, so animal husbandry industry has developed rapidly. However, due to the lack of scientific knowledge and wanting to make more money, the phenomenon that some farmers use veterinary drug illegally is widespread in the breeding industry, and the situation is particularly serious in China. In recent years, Chinese food exports were blocked repeatedly because of the problem of drug residues. It caused huge economic losses, and damaged the image of Chinese food seriously. Drug residues lead to not only human acute and chronic toxic directly and bacterial resistance, but also potential harm to human health indirectly through the food chain, such as allergy, deformity, mutation, cancer and so on. Therefore, the safety issue of drug residues in animal-origin food has been highly concerned in the world.
Food safety work is not only the need to the development of animal husbandry and Chinese foreign trade is also the need to the political and economic stability. Therefore, it becomes important to control drug residues level in animal-origin food.
Many developed countries attach great importance to monitoring of drug residue; they issued a series of regulations, drawn up detailed standards for drug residue limits, and strictly implement the monitoring, supervision and inspection of drug residue. In recent years, our government also has a high regard to this issue, strict residue standards and off-drug period have been formulated to limit and effectively control drug residues in animal-origin food. They take law, economy, administration and technology to monitoring, draw lessons from foreign advanced experience and technology, and screen a set of feasible detection method about drugs residues on the basis of our nationality.
At present the analytical techniques to detect drug residues mainly include physical and chemical detection methods (such as HPLC、LC-MS/MS), microbial detection method, immunosorbent assay (including ELISA, RIA, EIA, FIA, MAIA). Physical and chemical detection methods can be considered as the standard method to detect drug residues, because it is accurate, stable and reliable, but the equipment used in these methods is expensive, plus complex, time-consuming and laborious sample treatment procedure. So it is difficult to popularize. The results measured by the microbial detection methods are usually higher than the Maximum Residue Limits (MRLs). A rapid, sensitive and effective detection method must be established. Enzyme-linked immunoassay (including ELISA, CL-ELISA, and MAIA) overcomes the shortcomings of the methods mentioned above and is a rapid, sensitive, convenient, easy universal detection method. This paper aims at establishing chemiluminescence enzyme-linked immunoassay (CL-ELISA) to detect ractopamine, magnetic affinity immunoassay (MAIA) to detect chloramphenicol and enzyme-linked immunoassay (ELISA) to detect melamine.
For ractopamine, first, we synthesized the immunogen (Rac-cBSA) via the Para amino benzoic acid scheme on the basis of the structure of ractopamine. The immunogen was injected into New Zealand white rabbits and we obtained polyclonal antibodies to develop an immunoassay in this study. At the same time, we prepared coating antigen by the BDE scheme. Then using the indirect CL-ELISA, we obtained the results:The anti-ractopamine shows high sensitivity, with an IC50 value of 0.45 ppb toward ractopamine; the antibody has a detective limit of 0.001 ppb and a linear range of 5.0-405 ppb; with high specificity, there is no detectable cross-reactivity with the most other similar drugs. Recovery rates from the ractopamine in milk samples were in the range of 70-125%, while the intra-assay and inter-assay coefficients of variation were<12.0% and<13.0% respectively.
For Chloramphenicol, through the direct MAIA, we obtained the results:The anti-Chloramphenicol shows high sensitivity, with an IC50 value of 0.05 ppb toward Chloramphenicol; the antibody has a linear range of 0.005-5.0 ppb; with high specificity, there is only lower cross-reactivity with Chloramphenicol Succinate (6.25%)and no detectable cross-reactivity with the most other similar drugs. Recovery rates from the Chloramphenicol in milk samples were in the range of 85.2-113.7%, while the coefficients of variation were in the range of 9.9-22%.
For melamine, first, we synthesized the immunogen (MEL-TG) via the EDC scheme on the basis of the structure of melamine. The immunogen was injected into mice and we obtained monoclonal antibody to develop an immunoassay in this study. At the same time, we prepared coating antigen by the same scheme. Then using the indirect ELISA, we obtained the results:The anti-melamine shows high sensitivity, with an IC50 value of 22.6 ppb toward melamine; the antibody has a detective limit of 5.0 ppb and a linear range of 0.01-10.0 ppb; with high specificity, there is no detectable cross-reactivity with the most other similar drugs. Recovery rates from the ractopamine in milk samples were in the range of 78-120%, while the intra-assay and inter-assay coefficients of variation were<18.1% and<24.7% respectively.
Through this study, we have successfully prepared specific polyclonal antibody for ractopamine, and monoclonal antibody for melamine. Using these antibodies, we established rapid sensitive methods to detect the residues of ractopamine Chloramphenicol and melamine, at the same time, we identified CL-ELISA and MAIA shows a significant improvement in sensitivity and detestability compared with traditional ELISA method. There researches can be used to develop various local immunoassay kits and open the way for detection kits of other drugs
做好食品安全工作不仅是保障人类健康,促进畜牧业发展和我国对外贸易的需要,也是国家政治经济稳定的需要。控制动物源性食品中药物残留已经成为目前确保动物源性食品安全的一项重要措施,许多经济发达国家十分重视药物残留监控,颁布了一系列畜产品药物残留管理法规,制定了详尽的药物残留限量标准,严格实施对畜产品药物残留的监控、监督与检测。近年来,我国也高度重视这一问题,为了有效地控制动物源性产品中的药物残留,制定了严格的残留标准和休药期以限制违禁药物在食品中的残留,采取法律、经济、行政和技术手段实行监控,并借鉴国外先进经验和技术,结合我国国情,研究、筛选出一套可行的违禁药物在食品中的残留检测方法。
目前国内外采用的残留分析检测方法主要有理化检测法(包括HPLC、LC-MS、LC-MS/MS等仪器法)、微生物检测法、免疫检测法(包括ELISA、RIA、EIA、FIA、MAIA)等。理化检测法准确、稳定、可靠,可以作为标准方法,但仪器设备昂贵,样品前处理复杂,费时,费力,不易普及；微生物检测法往往会高出最高检测量(MRLs),灵敏度不高。因此必须建立快速、灵敏、有效的检测方法。免疫检测法中的酶联免疫检测法(包括ELISA、CL-ELISA、MAIA等)克服了以上方法的缺点,是一种快速、灵敏、方便、易普及的检测方法,有着广阔的发展前景。本论文旨在建立莱克多巴胺的化学发光酶联免疫检测法(CL-ELISA)、氯霉素的磁分离酶联免疫检测法(MATA)和三聚氰胺的酶联免疫检测法(ELISA)
在对莱克多巴胺的研究中,首先根据其结构特点,利用对氨基苯甲酸法合成了免疫原(Rac-cBSA),将此免疫原免疫新西兰大白兔制得莱克多巴胺的多克隆抗体,合成免疫原的同时,我们用BDE法合成了包被抗原(Rac-OVA)。然后利用间接CL-ELISA研究莱克多巴胺抗体的亲和度、灵敏度、交叉反应及在牛奶中莱克多巴胺回收率的测定。经检测,IC50是0.45 ppb,检测限为0.001 ppb,线性范围为0.01-10 ppb；该抗体对其它p-兴奋剂交叉反应率很小；莱克多巴胺在牛奶中的添加回收率在78-120%之间,批内变异系数低于18.1%,批间变异系数低于24.7%。
在对氯霉素的研究中,通过直接MAIA法测得氯霉素单克隆抗体的亲和度、灵敏度、交叉反应及在牛奶中氯霉素回收率的测定。经检测,氯霉素抗体的IC50是0.05ppb,线性范围为0.005-5 ppb；该抗体仅对琥珀氯霉素有交叉反应(交叉反应率为6.25%),对其它的酰胺醇类抗生素的交叉反应率很小；检测氯霉素在牛奶中的添加回收率在85.2-113.7%之间,变异系数在9.9-22%之间。
在对三聚氰胺的研究中,首先根据其结构特点,利用水溶性碳二亚胺法合成了免疫原(MEL-TG),将此免疫原免疫小鼠,经过细胞融合与克隆化得到三聚氰胺的单克隆抗体,合成免疫原的同时,我们用同样的方法合成了包被抗原(MEL-BSA)。然后利用间接ELISA法研究了三聚氰胺抗体的灵敏度、交叉反应及在牛奶中三聚氰胺回收率的测定。经检测,IC50是22.6 ppb,检测限为5.0 ppb,线性范围为5.0-405 ppb；该抗体对其它类似物交叉反应率很小；三聚氰胺在牛奶中的添加回收率在70-125%之间,批内变异系数低于12.0%,批间变异系数低于13.0%。
通过本课题的研究,成功制备了用于检测莱克多巴胺的多克隆抗体和检测三聚氰胺的单克隆抗体,利用这些抗体,本研究建立了莱克多巴胺的化学发光酶联免疫检测法、氯霉素的磁分离酶联免疫检测法和三聚氰胺的酶联免疫检测法,并且验证了化学发光酶联免疫法和磁分离酶联免疫检测法比起传统的比色酶联免疫检测法具有更好的灵敏度和检测下限,为最终开发商品化的国产化试剂盒奠定了良好的基础,并为其它药物抗体的制备和检测试剂盒的开发开辟了道路。
With the development of international trade in food, the need for the animal-origin food is increasing greatly, so animal husbandry industry has developed rapidly. However, due to the lack of scientific knowledge and wanting to make more money, the phenomenon that some farmers use veterinary drug illegally is widespread in the breeding industry, and the situation is particularly serious in China. In recent years, Chinese food exports were blocked repeatedly because of the problem of drug residues. It caused huge economic losses, and damaged the image of Chinese food seriously. Drug residues lead to not only human acute and chronic toxic directly and bacterial resistance, but also potential harm to human health indirectly through the food chain, such as allergy, deformity, mutation, cancer and so on. Therefore, the safety issue of drug residues in animal-origin food has been highly concerned in the world.
Food safety work is not only the need to the development of animal husbandry and Chinese foreign trade is also the need to the political and economic stability. Therefore, it becomes important to control drug residues level in animal-origin food.
Many developed countries attach great importance to monitoring of drug residue; they issued a series of regulations, drawn up detailed standards for drug residue limits, and strictly implement the monitoring, supervision and inspection of drug residue. In recent years, our government also has a high regard to this issue, strict residue standards and off-drug period have been formulated to limit and effectively control drug residues in animal-origin food. They take law, economy, administration and technology to monitoring, draw lessons from foreign advanced experience and technology, and screen a set of feasible detection method about drugs residues on the basis of our nationality.
At present the analytical techniques to detect drug residues mainly include physical and chemical detection methods (such as HPLC、LC-MS/MS), microbial detection method, immunosorbent assay (including ELISA, RIA, EIA, FIA, MAIA). Physical and chemical detection methods can be considered as the standard method to detect drug residues, because it is accurate, stable and reliable, but the equipment used in these methods is expensive, plus complex, time-consuming and laborious sample treatment procedure. So it is difficult to popularize. The results measured by the microbial detection methods are usually higher than the Maximum Residue Limits (MRLs). A rapid, sensitive and effective detection method must be established. Enzyme-linked immunoassay (including ELISA, CL-ELISA, and MAIA) overcomes the shortcomings of the methods mentioned above and is a rapid, sensitive, convenient, easy universal detection method. This paper aims at establishing chemiluminescence enzyme-linked immunoassay (CL-ELISA) to detect ractopamine, magnetic affinity immunoassay (MAIA) to detect chloramphenicol and enzyme-linked immunoassay (ELISA) to detect melamine.
For ractopamine, first, we synthesized the immunogen (Rac-cBSA) via the Para amino benzoic acid scheme on the basis of the structure of ractopamine. The immunogen was injected into New Zealand white rabbits and we obtained polyclonal antibodies to develop an immunoassay in this study. At the same time, we prepared coating antigen by the BDE scheme. Then using the indirect CL-ELISA, we obtained the results:The anti-ractopamine shows high sensitivity, with an IC50 value of 0.45 ppb toward ractopamine; the antibody has a detective limit of 0.001 ppb and a linear range of 5.0-405 ppb; with high specificity, there is no detectable cross-reactivity with the most other similar drugs. Recovery rates from the ractopamine in milk samples were in the range of 70-125%, while the intra-assay and inter-assay coefficients of variation were<12.0% and<13.0% respectively.
For Chloramphenicol, through the direct MAIA, we obtained the results:The anti-Chloramphenicol shows high sensitivity, with an IC50 value of 0.05 ppb toward Chloramphenicol; the antibody has a linear range of 0.005-5.0 ppb; with high specificity, there is only lower cross-reactivity with Chloramphenicol Succinate (6.25%)and no detectable cross-reactivity with the most other similar drugs. Recovery rates from the Chloramphenicol in milk samples were in the range of 85.2-113.7%, while the coefficients of variation were in the range of 9.9-22%.
For melamine, first, we synthesized the immunogen (MEL-TG) via the EDC scheme on the basis of the structure of melamine. The immunogen was injected into mice and we obtained monoclonal antibody to develop an immunoassay in this study. At the same time, we prepared coating antigen by the same scheme. Then using the indirect ELISA, we obtained the results:The anti-melamine shows high sensitivity, with an IC50 value of 22.6 ppb toward melamine; the antibody has a detective limit of 5.0 ppb and a linear range of 0.01-10.0 ppb; with high specificity, there is no detectable cross-reactivity with the most other similar drugs. Recovery rates from the ractopamine in milk samples were in the range of 78-120%, while the intra-assay and inter-assay coefficients of variation were<18.1% and<24.7% respectively.
Through this study, we have successfully prepared specific polyclonal antibody for ractopamine, and monoclonal antibody for melamine. Using these antibodies, we established rapid sensitive methods to detect the residues of ractopamine Chloramphenicol and melamine, at the same time, we identified CL-ELISA and MAIA shows a significant improvement in sensitivity and detestability compared with traditional ELISA method. There researches can be used to develop various local immunoassay kits and open the way for detection kits of other drugs
引文
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26. Henze M.K., Opfermann G., Spahn-Langguth H., Schanzer W. Screening of β-agonists and confirmation of fenoterol, orciprenaline, reproterol and terbutaline with gas chromatography-mass spectrometry as tetrahydroisoquinoline derivatives. J. Chromatogr. B.2001,751,93-105
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31. Shelver, Weilin L., Smith, David J. Determination of Ractopamine in Cattle and Sheep Urine Samples Using an Optical Biosensor Analysis:Comparative Study with HPLC and ELISA. J. Agric. Food Chem.2003,51,3715-3721
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41. Forti, A. F.; Campana, G.; Simonella, A.; Multari, M.; Scortichini, G. Determination of chloramphenicol in honey by liquid chromatography-tandem mass spectrometry. Anal. Chim. Acta.2005,529,257-263
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43. Gantverg, A., Shishani, I. and Hoffman, M. Determinayion of chloraphenicol in animal tissue and urine liquid chromatography-tandem mass spectrometry versus gas chromatography-mass spectrometry, Anal. Chem. Acta.2003,483,125-135
44. Santos, L. and Barbosa, J. Determination of chloramphenicol residues in rainbow trouts by gas chromatography-mass spectometry and liquid chromatography-tandem mass spectrometry, Anal. Chem. Acta.2005,529,249-256
45. Penney, L., Smith, A., Coates, B. and Wijewickreme, A. Determination of chloramphenicol residues in milk, eggs, and tissues by liquid chromatography/mass spectrometry,J. of AOAC Inter.2005,88,645-653.
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48. Takino, M.; Daishima, S.; Nakahara, T. Determination of chloramphenicol residues in fish meats by liquid chromatography-atmospheric pressure photoionization mass spectrometry. J. Chromatogr. A 2003,1011,67-75.
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52. Ashwin, H.M.; Stead, S.L; Taylor, J.C.; Martin, J.R.; Richmond, S.F.; Homer, V.; Bigwood, T.; Sharman, M. Development and validation of screening and confirmatory methods forthe detection of chloramphenicol and chloramphenicol glucuronide using SPR biosensor and liquid chromatography-tandem mass spectrometry Anal. Chim. Acta. 2005,529,103-108
53. In-Seon Park; Namsoo Kim. Development of a chemiluminescent immunosensor for Chloramphenicol. Analytica Chimica Acta.2006,578,19-24
54. Gaudin V.; Maris P. Development of a Biosensor-based Immunoassay for Screening of Chloramphenicol Residues in Milk. Food and Agricultural Immunology.2001,13, 77-86
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56. Nan Liu, Pu Su, Zhixian Gao, Maoxiang Zhu, Zhihua Yang, Xiujie Pan, Yanjun Fang. Fuhuan Chao. Simultaneous detection for three kinds of veterinary drugs: Chloramphenicol, clenbuterol and 17-beta-estradiol by high-throughput suspension array technology. Analytica Chimica Acta.2009,632,128-134
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