首页>>学术检索

畜禽饲料中硝基呋喃类药物快速检测技术研究

标题: 畜禽饲料中硝基呋喃类药物快速检测技术研究
英文标题: Study on the Methods for Rapid Determination of Nitrofurans in Animal Feeds
作者: 李军
出版时间: 2011-01-01
所在大学: 河北农业大学
关键词: 5-硝基糠醛,硝基呋喃类药物,广谱特异性抗体,ELISA,HPLC
英文关键词: nitrofuran,5-nitrofurfural,Broad specific antibody,ELISA,HPLC
论文级别: 硕士
学位: 学位论文
导师: 王建平
专业: 基础兽医学
提交时间: 2011
摘要: 本研究以5-硝基糠醛为分子模板合成了硝基呋喃类药物的共有半抗原,分别采用重氮法和戊二醛法合成了两种免疫抗原和两种包被抗原.重氮法合成的免疫原的偶联比为13:1,包被原偶联比为14:1;戊二醛法合成的免疫原的偶联比为20:1,包被原偶联比为16:1.用两种免疫抗原免疫新西兰白兔,所得抗血清经辛酸-硫酸铵法沉淀和DEAE-纤维素阴离子交换层析纯化,得到纯度较高的抗体IgG.以方阵滴定法确定重氮法免疫原所得抗体的效价为1:320 000,戊二醛法免疫原所得抗体的效价为1:640 000.两种抗体均对7种硝基呋喃类药物具有广谱交叉反应性.在优化了不同包被原与不同抗体的组合后,以重氮法合成的包被原和戊二醛法合成的免疫原所得抗体为基础建立了一种检测7种硝基呋喃类药物的广谱特异性间接竞争ELISA方法.该法对呋喃西林、硝呋酚酰肼、硝呋索尔、呋喃唑酮、呋喃它酮、呋喃妥因、呋喃苯烯酸钠的交叉反应率分别为95%、79%、76%、68%、61%、59%、42%,IC50分别为95、79、76、68、61、59、42 ng/mL,最低检测限分别为8.3、14.0、15.8、11.0、11.0、13.5、18.2 ng/mL.在空白全价饲料中分别添加7种硝基呋喃类药物,以ELISA方法检测,添加回收率范围为78.0%-98.4%,变异系数为6.2%-13.8%.同时,建立了一种检测7种硝基呋喃类药物的HPLC方法,7种药物在0.01-1μg/mL范围内有良好的线形关系.该法对全价饲料中呋喃西林、呋喃它酮、呋喃唑酮、硝呋酚酰肼、呋喃苯烯酸钠、呋喃妥因、硝呋索尔的检测限分别为21、26、28、32、17、19、20 ng/g,在空白全价饲料中的添加回收率为70.1-99.6%.本研究建立了一种快速、灵敏的检测饲料中7种硝基呋喃类药物的ELISA方法,并以一种高效液相色谱法给予确证,以期能更加快速有效的监控养殖业中硝基呋喃类药物的非法使用,保障动物饲料及动物性产品安全.
英文摘要: The genetic hapten of nitrofurans was synthesized with 5-nitrofurfural (NFF) as the template. Then two immunogens and two coating antigens were prepared by use of diazotization method and glutaraldehyde method, respectively. The coupling ratios of NFF to BSA in two immunogens were 13:1 and 20:l, respectively, and those of to OA in two coating antigens were 14:1 and 16:1, respectively. The two immunogens were used to immunize New Zealand white rabbits to produce the antibodies. The antisera were purified by acrylic acid-ammonium sulfate precipitation method and DEAE-cellulose anion-exchange chromatography to obtain the IgG fraction. The checkboard titration method was used to determine the antibody titer from diazotization method as 1: 320000 and the antibody titer from glutaraldehyde method was 1:640000. Both of the two antibodies showed broad specificity to seven nitrofurans (nitrofurazone, nifuroxazide, nifursol, furazolidone, furaltadone, and nitrofurantoin and nifurstyrenate sodium). After optimization of different coating antigen-antibody combinations, the coating antigen from diazotization method and the antibody from glutaraldehyde method were used to develop an indirect competitive enzyme linked immunosorbent assay (ELISA). The cross-reactivity to the seven nitrofurans were 95%、79%、76%、68%、61%、59%、42%, respectively, the IC50 values were 95、79、76、68、61、59、42ng/mL, respectively, and the limits of detection were 8.3、14.0、15.8、11.0、11.0、13.5、18.2ng/mL, respectively. The seven nitrofuran drugs were fortified into blank feeds at different levels and the recoveries were in a range of 78.0%-98.4%with coefficients of variation of 6.2%-13.8%. Furthermore, a HPLC method was developed to determine the 7 nitrofuran drugs. These drugs were in good linearity in the range of 0.01-1μg/mL. The limits of detection for the seven drugs in feeds were 21, 26, 28, 32, 17, 19, 20ng/g, respectively and the recoveries were in a range of 70.1%-99.6%.In this study, a rapid and sensitive ELISA method was developed to detect seven nitrofurans in animal feeds simultaneously and a HPLC method was established as the confirmatory method. The two methods could be used to determine the presence of nitrofuran drugs in animal feeds to monitor the illicit use of nitrofuran in animal husbandry and ensure the safety of animal feeds and animal-original food.