呋喃妥因和呋喃唑酮在食品中残留的酶联免疫测定法的研究
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摘要
随着经济的迅速发展,人民的生活水平不断提高,对肉蛋类产品的需求量大大增加,因而现代的养殖业得到了迅速发展。硝基呋喃类药物是人工合成的广谱抗生素,因其价格较低且治疗效果好,一些经营者为追求经济利益,而广泛用于畜禽及水产养殖业,并作为治疗药物和饲料添加剂。但是多年来的研究表明,硝基呋喃类药物及其代谢产物具有致癌、致畸、致突变性,这些药物的大量使用不可避免的残留于动物的肉、蛋、奶、脏器、组织中,从而影响人体健康,所以世界上许多国家,比如欧盟、美国、中国、日本等都严格控制硝基呋喃类药物用于畜禽及水产养殖业。因此食品安全问题已引起全世界的高度重视。
现在很多国家已经建立了一些药物残留的检测方法。传统检测硝基呋喃类药物残留的分析方法包括HPLC、LC-UV、LC-MS、LC-MS-MS等仪器法。这些方法准确、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,因此必须建立快速、灵敏、有效的检测方法。酶联免疫检测法(ELISA)克服了以上方法的缺点,是一种快速、灵敏、方便的检测方法,有着广阔的发展前景。因此,本论文旨在建立呋喃妥因和呋喃唑酮及其代谢产物的酶联免疫吸附检测法(ELISA)。
对于呋喃妥因及其代谢产物AHD免疫原的制备,根据其结构特点,从其代谢产物(AHD)出发,采用多种方案与载体蛋白偶联,通过免疫只有混合酸酐法合成的免疫原是成功的。它是利用AHD先与CBA偶联引入一羧基,然后利用混合酸酐法与载体蛋白合成了免疫原。合成免疫原的同时,也利用相同的方法合成了多种包被抗原。利用混合酸酐法合成的免疫原免疫新西兰白兔获得了能同时检测呋喃妥因和AHD(以衍生物NPAHD的形式存在)的抗体,得到的抗体分别与多种包被抗原进行交叉测定,结果利用戊二醛法合成的包被抗原进行检测灵敏性及特异性较好。对呋喃妥因抗体其检测结果为:IC_(50)是3.2ng/mL,检测限为0.2 ng/mL。该抗体除对CPAHD和NPAHD的交叉反应率较高外,和其他药物的交叉率均较小。呋喃妥因在养殖用水中的添加回收率批内在90-103%之间,批间在88-103%之间。批内变异系数在2.7-6.2%之间,批间变异系数在3.1-11.4%之间。对AHD抗体其检测结果为:IC_(50)(转化为AHD计算)是10.6 ng/mL,检测限为0.05 ng/mL。该抗体除对CPAHD和呋喃妥因的交叉反应率较高外,和其他药物的交叉率均较小。AHD在猪肝中的添加回收率批内在91-104%之间,批间在80-96%之间。批内变异系数在2.4-17.7%之间,批间变异系数在3.7-14.6%之间。
对呋喃唑酮及其代谢产物AOZ的研究,其免疫原和包被抗原的合成方法,与呋喃妥因及其代谢产物的相似,也是从代谢产物AOZ出发,利用多种方案合成了免疫原,通过检测免疫后得到的抗体,用混合酸酐法合成的免疫原免疫得到的抗体,用戊二醛法合成的包被抗原进行检测结果较好,免疫得到的抗体可以同时检测呋喃唑酮及其代谢产物AOZ。对呋喃唑酮的抗体其检测结果为:IC_(50)是2.9 ng/mL,检测限为0.2 ng/mL。该抗体除与CPAOZ和NPAOZ的交叉反应率较高外,和其他药物的交叉率均较小。呋喃唑酮在养殖用水中的添加回收率批内在76-92%之间,批间在74-88%之间。批内变异系数在3.8-14%之间,批间变异系数在3.4-9.2%之间。对AOZ抗体的检测,我们只做了灵敏性及特异性的分析,其检测结果为:IC_(50)(转化为AOZ计算)是3.9 ng/mL,检测限为0.18ng/mL。该抗体除对CPAOZ和呋喃唑酮的交叉反应率较高外,和其他药物的交叉率均较小。
通过对硝基呋喃类药物的研究,我们成功地制备了呋喃妥因和呋喃唑酮及其代谢产物的免疫原和包被抗原,并通过动物免疫制备了相应的多克隆抗体,建立了呋喃妥因和呋喃唑酮及其代谢产物的酶联免疫检测法,并用于实际体系检测,检测结果显示该方法的重复性、精确度、准确度较好。
With the development of society and the improvement of life level,the need for the animal food is increasing rapidly.Therefore,modern animal husbandry is developed very rapidly in recent years.To make more money,many farmers use some drugs in animal husbandry.Nitrofurans as a group of synthetic antibiotics are used widely to treat animal diseases or as feed additives.But long-term research showed that the nitrofurans and their metabolites showed carcinogenic、abnormal and mutagenic characteristics,so the illegal use of these drugs in animal husbandry is inevitable to result in the presence of residues in edible tissues and can have potential health risks to human.Therefore,Nitrofurans are controlled strictly used in aquiculture and poultry in many countries,such as Europe,America,China and Japan. So the safety issue of food has been highly concerned in the world.
To ensure the safety of food for consumers,many countries have established analytical techniques to detect residues of drugs.The traditionally instrumental analyses such as HPLC、LC-UV、LC-MS and LC-MS-MS are the most widely used methods to detect residues of drugs in food and food products.These methods are sensitive,accurate and highly specific,but require expensive equipment,large volume of solvents,derivative treatment,and time-consuming sample clean-up process. Therefore,they are not suitable to be used as routing screen and field detection methods for drugs.To detect residue of drugs in food and food products,ELISA is a low cost,high volume screening methods.So we can combine ELISA methods with instrumental analyses to ensure efficient and cost-effective use of resources.The aim of this research is to establish the ELISA techniques to detect the residues of nitrofurantoin and furazolidone and their metabolites AOZ and AHD.
To develop a rapid and convenient detection method to measure the residue of nitrofurantoin and AHD,we designed several immunogens via different methods on the basis of the structures of nitrofurantoin and AHD and prepared polyclonal antibodies to develop an immunoassay in this study.At the same time,we prepared coating antigen by the same methods as the preparation of the immunogen.The antibody obtained via acid anhydride scheme which is detected through the coating antigen prepared by glutaricdialdehyde method is successful.It can detect nitrofurantoin and AHD(in the form of NPAHD)at one time.The immunoassay based on the antibody for nitrofurantoin showed excellent specificity and sensitivity with IC_(50)of 3.2ng/mL and no detectable cross reaction with most related species and compounds except with CPAHD and NPAHD.The limit of detection,defined as IC_(10), was 0.2ng/mL.Considering that nitrofurantoin is often used illegally to feed animals through drinking water,we measured the residue of nitrofurantoin in water spiked by the drug.The recovery rates are in the range of 88-103%for inter-assay and 90-103% for intra-assay.The CVs are in the range of 3.1-11.4%for inter-assay and 2.7-6.2% for intra-assay.The immunoassay based on the antibody for AHD showed excellent specificity and sensitivity with IC_(50)(equivalent underivatised AHD)is 10.6ng/mL and no detectable cross reaction with nitrofurans and other nitrofuran metabolites except with CPAHD and nitrofurantoin.The limit of detection was 0.05ng/mL(equivalent underivatised AHD).For the residue of AHD in swine liver,the recovery rates are in the range of 80-96%for inter-assay and 91-104%for intra-assay.The CVs are in the range of 2.4-17.7%for inter-assay and 3.7-14.6%for intra-assay.
To develop an antibody that is specific to furazolidone and AOZ,we designed the immunogen and coating antigen by the similar methods as the preparation of the immunogen and coating antigen of nitrofurantoin and AHD.The antibody obtained via acid anhydride scheme which is detected through the coating antigen prepared by glutaricdialdehyde method is successful.It can detect furazolidone and AOZ(in the form of NPAOZ)at one time.For the residue of furazolidone,the immunoassay based on the antibody showed excellent specificity and sensitivity with IC_(50)of 2.9ng/mL and no detectable cross reaction with most related species and compounds except with CPAOZ and NPAOZ.The limit of detection was below 0.2ng/mL.We measured the residue of furazolidone in water spiked by the drug.The recovery rates are in the range of 74-88%for inter-assay and 76-92%for intra-assay.The CVs are in the range of 3.8-14%for inter-assay and 3.4-9.2%for intra-assay.The immunoassay based on the antibody for AOZ showed excellent specificity and sensitivity with IC_(50)of 3.9ng/mL and no detectable cross reaction with most related species and compounds except with CPAOZ and furazolidone.The limit of detection was about 0.18ng/mL (equivalent underivatised AOZ).
In conclusion,we have successfully prepared the immunogens and coating antigens for nitrofurantoin and furazolidone as well as their metabolites,and we developed immunoassays based on the antibodies prepared successfully.The indirect ELISA methods developed in this study can be used as screen methods to detect residues in foods and food products.
现在很多国家已经建立了一些药物残留的检测方法。传统检测硝基呋喃类药物残留的分析方法包括HPLC、LC-UV、LC-MS、LC-MS-MS等仪器法。这些方法准确、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,因此必须建立快速、灵敏、有效的检测方法。酶联免疫检测法(ELISA)克服了以上方法的缺点,是一种快速、灵敏、方便的检测方法,有着广阔的发展前景。因此,本论文旨在建立呋喃妥因和呋喃唑酮及其代谢产物的酶联免疫吸附检测法(ELISA)。
对于呋喃妥因及其代谢产物AHD免疫原的制备,根据其结构特点,从其代谢产物(AHD)出发,采用多种方案与载体蛋白偶联,通过免疫只有混合酸酐法合成的免疫原是成功的。它是利用AHD先与CBA偶联引入一羧基,然后利用混合酸酐法与载体蛋白合成了免疫原。合成免疫原的同时,也利用相同的方法合成了多种包被抗原。利用混合酸酐法合成的免疫原免疫新西兰白兔获得了能同时检测呋喃妥因和AHD(以衍生物NPAHD的形式存在)的抗体,得到的抗体分别与多种包被抗原进行交叉测定,结果利用戊二醛法合成的包被抗原进行检测灵敏性及特异性较好。对呋喃妥因抗体其检测结果为:IC_(50)是3.2ng/mL,检测限为0.2 ng/mL。该抗体除对CPAHD和NPAHD的交叉反应率较高外,和其他药物的交叉率均较小。呋喃妥因在养殖用水中的添加回收率批内在90-103%之间,批间在88-103%之间。批内变异系数在2.7-6.2%之间,批间变异系数在3.1-11.4%之间。对AHD抗体其检测结果为:IC_(50)(转化为AHD计算)是10.6 ng/mL,检测限为0.05 ng/mL。该抗体除对CPAHD和呋喃妥因的交叉反应率较高外,和其他药物的交叉率均较小。AHD在猪肝中的添加回收率批内在91-104%之间,批间在80-96%之间。批内变异系数在2.4-17.7%之间,批间变异系数在3.7-14.6%之间。
对呋喃唑酮及其代谢产物AOZ的研究,其免疫原和包被抗原的合成方法,与呋喃妥因及其代谢产物的相似,也是从代谢产物AOZ出发,利用多种方案合成了免疫原,通过检测免疫后得到的抗体,用混合酸酐法合成的免疫原免疫得到的抗体,用戊二醛法合成的包被抗原进行检测结果较好,免疫得到的抗体可以同时检测呋喃唑酮及其代谢产物AOZ。对呋喃唑酮的抗体其检测结果为:IC_(50)是2.9 ng/mL,检测限为0.2 ng/mL。该抗体除与CPAOZ和NPAOZ的交叉反应率较高外,和其他药物的交叉率均较小。呋喃唑酮在养殖用水中的添加回收率批内在76-92%之间,批间在74-88%之间。批内变异系数在3.8-14%之间,批间变异系数在3.4-9.2%之间。对AOZ抗体的检测,我们只做了灵敏性及特异性的分析,其检测结果为:IC_(50)(转化为AOZ计算)是3.9 ng/mL,检测限为0.18ng/mL。该抗体除对CPAOZ和呋喃唑酮的交叉反应率较高外,和其他药物的交叉率均较小。
通过对硝基呋喃类药物的研究,我们成功地制备了呋喃妥因和呋喃唑酮及其代谢产物的免疫原和包被抗原,并通过动物免疫制备了相应的多克隆抗体,建立了呋喃妥因和呋喃唑酮及其代谢产物的酶联免疫检测法,并用于实际体系检测,检测结果显示该方法的重复性、精确度、准确度较好。
With the development of society and the improvement of life level,the need for the animal food is increasing rapidly.Therefore,modern animal husbandry is developed very rapidly in recent years.To make more money,many farmers use some drugs in animal husbandry.Nitrofurans as a group of synthetic antibiotics are used widely to treat animal diseases or as feed additives.But long-term research showed that the nitrofurans and their metabolites showed carcinogenic、abnormal and mutagenic characteristics,so the illegal use of these drugs in animal husbandry is inevitable to result in the presence of residues in edible tissues and can have potential health risks to human.Therefore,Nitrofurans are controlled strictly used in aquiculture and poultry in many countries,such as Europe,America,China and Japan. So the safety issue of food has been highly concerned in the world.
To ensure the safety of food for consumers,many countries have established analytical techniques to detect residues of drugs.The traditionally instrumental analyses such as HPLC、LC-UV、LC-MS and LC-MS-MS are the most widely used methods to detect residues of drugs in food and food products.These methods are sensitive,accurate and highly specific,but require expensive equipment,large volume of solvents,derivative treatment,and time-consuming sample clean-up process. Therefore,they are not suitable to be used as routing screen and field detection methods for drugs.To detect residue of drugs in food and food products,ELISA is a low cost,high volume screening methods.So we can combine ELISA methods with instrumental analyses to ensure efficient and cost-effective use of resources.The aim of this research is to establish the ELISA techniques to detect the residues of nitrofurantoin and furazolidone and their metabolites AOZ and AHD.
To develop a rapid and convenient detection method to measure the residue of nitrofurantoin and AHD,we designed several immunogens via different methods on the basis of the structures of nitrofurantoin and AHD and prepared polyclonal antibodies to develop an immunoassay in this study.At the same time,we prepared coating antigen by the same methods as the preparation of the immunogen.The antibody obtained via acid anhydride scheme which is detected through the coating antigen prepared by glutaricdialdehyde method is successful.It can detect nitrofurantoin and AHD(in the form of NPAHD)at one time.The immunoassay based on the antibody for nitrofurantoin showed excellent specificity and sensitivity with IC_(50)of 3.2ng/mL and no detectable cross reaction with most related species and compounds except with CPAHD and NPAHD.The limit of detection,defined as IC_(10), was 0.2ng/mL.Considering that nitrofurantoin is often used illegally to feed animals through drinking water,we measured the residue of nitrofurantoin in water spiked by the drug.The recovery rates are in the range of 88-103%for inter-assay and 90-103% for intra-assay.The CVs are in the range of 3.1-11.4%for inter-assay and 2.7-6.2% for intra-assay.The immunoassay based on the antibody for AHD showed excellent specificity and sensitivity with IC_(50)(equivalent underivatised AHD)is 10.6ng/mL and no detectable cross reaction with nitrofurans and other nitrofuran metabolites except with CPAHD and nitrofurantoin.The limit of detection was 0.05ng/mL(equivalent underivatised AHD).For the residue of AHD in swine liver,the recovery rates are in the range of 80-96%for inter-assay and 91-104%for intra-assay.The CVs are in the range of 2.4-17.7%for inter-assay and 3.7-14.6%for intra-assay.
To develop an antibody that is specific to furazolidone and AOZ,we designed the immunogen and coating antigen by the similar methods as the preparation of the immunogen and coating antigen of nitrofurantoin and AHD.The antibody obtained via acid anhydride scheme which is detected through the coating antigen prepared by glutaricdialdehyde method is successful.It can detect furazolidone and AOZ(in the form of NPAOZ)at one time.For the residue of furazolidone,the immunoassay based on the antibody showed excellent specificity and sensitivity with IC_(50)of 2.9ng/mL and no detectable cross reaction with most related species and compounds except with CPAOZ and NPAOZ.The limit of detection was below 0.2ng/mL.We measured the residue of furazolidone in water spiked by the drug.The recovery rates are in the range of 74-88%for inter-assay and 76-92%for intra-assay.The CVs are in the range of 3.8-14%for inter-assay and 3.4-9.2%for intra-assay.The immunoassay based on the antibody for AOZ showed excellent specificity and sensitivity with IC_(50)of 3.9ng/mL and no detectable cross reaction with most related species and compounds except with CPAOZ and furazolidone.The limit of detection was about 0.18ng/mL (equivalent underivatised AOZ).
In conclusion,we have successfully prepared the immunogens and coating antigens for nitrofurantoin and furazolidone as well as their metabolites,and we developed immunoassays based on the antibodies prepared successfully.The indirect ELISA methods developed in this study can be used as screen methods to detect residues in foods and food products.
引文
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50. Kim, Y. J.; Cho, Y. A.; Lee, H. S.; Lee, Y. T.; Gee, S. J.; Hammock, B.D. Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity. Anal. Chim. Acta. 2003, 475, 85-96
51. Szurdoki, R; Szekacs, A.; Le, H. M; Hammock, B. D. Synthesis of haptens and protein conjugates for the development of immunoassays for the insect growth regulator fenoxycarb. J. Agric. Food Chem. 2002, 50, 29-40