呋喃它酮和呋喃妥因酶联免疫检测方法的研究

发布时间：2018-10-25 12:38

【摘要】：本论文对动物源性食品中硝基呋喃类抗生素呋喃它酮和呋喃妥因及其代谢物的酶联免疫检测方法进行了系统研究。对于呋喃它酮,从其代谢物(AMOZ)出发,先将AMOZ与对醛基苯甲酸偶联,衍生为CPAMOZ,然后利用活化酯法将其与牛血清蛋白偶联制备免疫原,通过免疫新西兰大耳白兔得到高特异性和高灵敏度的多克隆抗体。利用戊二醛法将AMOZ与鸡卵白蛋白偶联制备包被原。通过优化包被抗原量、抗体稀释倍数、封闭液、离子强度和缓冲液pH,建立了异源间接竞争ELISA。该方法对NPAMOZ的IC50为0.47±0.08ng/mL,检出限(IC15)为0.04±0.008 ng/mL o利用活化酯法将CPAMOZ和辣根过氧化物酶偶联制备酶标抗原。通过优化抗体包被量、酶标抗原稀释倍数、封闭液、离子强度、缓冲液pH,建立了同源直接竞争ELISA。该方法对NPAMOZ的IC50为0.92±0.12 ng/mL,检出限(IC15)为0.1±0.08 ng/mLo抗体具有很好的特异性,除对呋喃它酮原药有交叉反应外,与其他硝基呋喃类药物及其代谢物的均没有交叉。选择鸡肉、鸡肝、牛肉、猪肉、虾、鲷鱼、木棉鱼、鱿鱼样品进行添加回收实验,样品被分别添加了浓度为0.2 ng/g、0.5 ng/g、1.0 ng/g、5.0 ng/g的AMOZ,经过过夜衍生后应用所建立的ELISA方法检测,回收率在74.3%-111.5%之间,且批内和批间的变异系数(n=3)小于20%。对添加回收结果进行方差分析,结果表明所建立的ELISA方法针对不同类别的动物组织的检测结果没有显著差异,方法具有很好的准确性和广泛的应用性。对于呋喃妥因从其代谢物(AHD)出发,先将AHD与对醛基苯甲酸偶联,衍生为CPAHD,利用混合酸酐法与牛血清偶联原制备免疫原,通过免疫新西兰大耳白兔得到高特异性和高灵敏度的多克隆抗体。利用活化酯法将CPAHD和辣根过氧化物酶偶联制备酶标抗原,通过优化抗体包被量、酶标抗原稀释倍数、封闭液、离子强度、缓冲液pH,建立了同源直接竞争ELISA。该方法对NPAHD的IC50为5.9±1.2 ng/mL,检出限(IC15) 0.51±0.12 ng/mL。抗体具有很好的特异性,除对呋喃妥因原药有交叉反应外,与其他硝基呋喃类药物及其代谢物的均没有交叉。选择鸡肉、牛肉、猪肉、虾、木棉鱼进行添加回收实验,样品被分别添加了浓度为2.0 ng/g、4.0 ng/g、20 ng/g、100 ng/g的AHD,经过过夜衍生后应用所建立的ELISA方法检测,回收率在71.7-104.1%之间,且批间和批内变异系数(n=3)小于20%。
[Abstract]:In this paper, enzyme linked immunosorbent assay (Elisa) for the detection of nitrofurantoin and furantoin and their metabolites in animal food was studied. For furantadone, starting from its metabolite (AMOZ), AMOZ was first coupled with p-aldobenzoic acid, then derived into CPAMOZ, and then it was coupled with bovine serum protein by activation ester method to prepare immunogen. High specific and sensitive polyclonal antibodies were obtained by immunizing New Zealand rabbits. The coating material was prepared by coupling AMOZ with chicken ovalbumin by glutaraldehyde method. The heterogenous indirect competitive ELISA. was established by optimizing the coating antigen volume, antibody dilution multiple, blocking solution, ionic strength and buffer solution pH,. The IC50 of NPAMOZ was 0. 47 卤0. 08 ng / mL, the detection limit (IC15) was 0. 04 卤0.008 ng/mL o. The enzyme labeled antigen was prepared by coupling CPAMOZ with horseradish peroxidase by activation ester method. The homologous direct competition ELISA. was established by optimizing antibody coating, dilution times of enzyme labeled antigen, blocking solution, ionic strength and buffer solution pH,. The detection limit (IC15) of IC50 of NPAMOZ was 0.92 卤0.12 ng/mL, and the detection limit (IC15) was 0.1 卤0.08 ng/mLo antibody. The method had no cross reaction with other nitrofurans and their metabolites except for the cross-reaction of furantadone. The samples of chicken, chicken liver, beef, pork, shrimp, snapper, kapok and squid were tested by adding 0.2 ng/g,0.5 ng/g,1.0 ng/g,5.0 ng/g AMOZ, after overnight derivatization. The recovery was between 74.3% and 111.5%, and the coefficient of variation (nc3) was less than 20.3%. The results of ANOVA show that there is no significant difference in the results of the ELISA method for different animal tissues, and the method has good accuracy and wide application. For furantoin, starting from its metabolite (AHD), AHD was first coupled with p-aldehyde benzoic acid, and then CPAHD, was derived to prepare immunogen by mixed anhydride method and bovine serum conjugation. High specific and sensitive polyclonal antibodies were obtained by immunizing New Zealand rabbits. CPAHD was coupled with horseradish peroxidase to prepare enzyme labeled antigen by activation ester method. The homologous and direct competitive ELISA. was established by optimizing antibody encapsulation, dilution times of enzyme labeled antigen, blocking solution, ionic strength and buffer pH,. The IC50 of this method for NPAHD is 5.9 卤1.2 ng/mL, detection limit (IC15) 0.51 卤0.12 ng/mL.. The antibody has good specificity, except for the cross-reaction of nitrofurantoin, it has no cross reaction with other nitrofuran drugs and their metabolites. Chicken, beef, pork, shrimp and kapok fish were selected for the recovery experiment. The samples were determined by the ELISA method with the concentration of 2.0 ng/g,4.0 ng/g,20 ng/g,100 ng/g after overnight derivatization. The recovery rate was 71.7-104.1%. The coefficient of variation (N3) between and within batches was less than 20.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R155.5

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