【摘要】：本論文對動物源性食品中硝基呋喃類抗生素呋喃它酮和呋喃妥因及其代謝物的酶聯(lián)免疫檢測方法進行了系統(tǒng)研究。對于呋喃它酮,從其代謝物(AMOZ)出發(fā),先將AMOZ與對醛基苯甲酸偶聯(lián),衍生為CPAMOZ,然后利用活化酯法將其與牛血清蛋白偶聯(lián)制備免疫原,通過免疫新西蘭大耳白兔得到高特異性和高靈敏度的多克隆抗體。利用戊二醛法將AMOZ與雞卵白蛋白偶聯(lián)制備包被原。通過優(yōu)化包被抗原量、抗體稀釋倍數(shù)、封閉液、離子強度和緩沖液pH,建立了異源間接競爭ELISA。該方法對NPAMOZ的IC50為0.47±0.08ng/mL,檢出限(IC15)為0.04±0.008 ng/mL o利用活化酯法將CPAMOZ和辣根過氧化物酶偶聯(lián)制備酶標抗原。通過優(yōu)化抗體包被量、酶標抗原稀釋倍數(shù)、封閉液、離子強度、緩沖液pH,建立了同源直接競爭ELISA。該方法對NPAMOZ的IC50為0.92±0.12 ng/mL,檢出限(IC15)為0.1±0.08 ng/mLo抗體具有很好的特異性,除對呋喃它酮原藥有交叉反應外,與其他硝基呋喃類藥物及其代謝物的均沒有交叉。選擇雞肉、雞肝、牛肉、豬肉、蝦、鯛魚、木棉魚、魷魚樣品進行添加回收實驗,樣品被分別添加了濃度為0.2 ng/g、0.5 ng/g、1.0 ng/g、5.0 ng/g的AMOZ,經(jīng)過過夜衍生后應用所建立的ELISA方法檢測,回收率在74.3%-111.5%之間,且批內(nèi)和批間的變異系數(shù)(n=3)小于20%。對添加回收結果進行方差分析,結果表明所建立的ELISA方法針對不同類別的動物組織的檢測結果沒有顯著差異,方法具有很好的準確性和廣泛的應用性。對于呋喃妥因從其代謝物(AHD)出發(fā),先將AHD與對醛基苯甲酸偶聯(lián),衍生為CPAHD,利用混合酸酐法與牛血清偶聯(lián)原制備免疫原,通過免疫新西蘭大耳白兔得到高特異性和高靈敏度的多克隆抗體。利用活化酯法將CPAHD和辣根過氧化物酶偶聯(lián)制備酶標抗原,通過優(yōu)化抗體包被量、酶標抗原稀釋倍數(shù)、封閉液、離子強度、緩沖液pH,建立了同源直接競爭ELISA。該方法對NPAHD的IC50為5.9±1.2 ng/mL,檢出限(IC15) 0.51±0.12 ng/mL�？贵w具有很好的特異性,除對呋喃妥因原藥有交叉反應外,與其他硝基呋喃類藥物及其代謝物的均沒有交叉。選擇雞肉、牛肉、豬肉、蝦、木棉魚進行添加回收實驗,樣品被分別添加了濃度為2.0 ng/g、4.0 ng/g、20 ng/g、100 ng/g的AHD,經(jīng)過過夜衍生后應用所建立的ELISA方法檢測,回收率在71.7-104.1%之間,且批間和批內(nèi)變異系數(shù)(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 鹵0. 08 ng / mL, the detection limit (IC15) was 0. 04 鹵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 鹵0.12 ng/mL, and the detection limit (IC15) was 0.1 鹵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 鹵1.2 ng/mL, detection limit (IC15) 0.51 鹵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.
【學位授予單位】：天津科技大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R155.5

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