食品中百草枯残留免疫学分析方法的研究与应用
摘要
百草枯(paraquat,PQ)是一种广谱、灭生性有机杂环类除草剂。由于百草枯的广泛应用,造成食品原料和水体污染,对人体健康产生潜在危害。目前百草枯检测方法大多需要专门的仪器设备并且操作繁琐,已无法满足现场快速检测的要求。因此,研究和建立一种方便、安全和快速的检测方法具有重要意义。本文以多克隆抗体和胶体金合成标记技术为基础,建立了百草枯的酶联免疫(ELISA)和金标免疫层析(GICA)检测方法,并将其应用于食品中的检测。
首先以4,4’-联吡啶和碘甲烷为起始原料,经三步反应合成了百草枯半抗原N-甲基-N′-戊酸基-二吡啶二溴化物,经LC/MS、1HNMR和IR鉴定结果表明,所得产物即为百草枯半抗原(简称PQ-h),其纯度为96%,得率为69%。通过混合酸酐法偶联大分子蛋白载体牛血清白蛋白(BSA)和卵清蛋白(OVA)制备免疫抗原(PQ-h-BSA)和包被抗原(PQ-h-OVA),并进行结构鉴定。利用分光光度法测定百草枯半抗原与BSA的偶联比为6:1。
通过免疫新西兰大白兔,获得特异性多克隆抗体,效价为51200。建立了PQ检测的间接竞争ELISA方法,经过多次棋盘试验,最终确立最适包被抗原浓度为0.3μg/mL,抗体最佳稀释度为1:5000。该方法的半数抑制率IC50为5.13 ng/mL,最低检出限为0.005 ng/mL。百草枯与敌草快的交叉反应率小于0.1%。大豆样品的加标回收率在70%~80%之间,相对标准偏差(RSD)低于10%。
采用柠檬酸三钠还原法制备胶体金,通过研究胶体金与抗体蛋白作用过程,确定稳定标记1 ml胶体金需8.1μg抗体蛋白,标记体系的最佳pH为8.5。以金标抗体作为分析探针,PQ-h-OVA作为竞争抗原,以羊抗兔IgG为控制抗体,构建直接竞争胶体金标免疫层析检测体系。确定膜上包被抗原和羊抗兔二抗的最佳包被浓度分别为0.5 mg/ml和0.11 mg/ml。金标目测检出限为10 ng/ml,检测时间约5 min。方法的重复性和稳定性较好,经推测,该试纸条在室温下至少可以保存5个月。对大豆进行加标回收试验,回收率较好。
本研究所建立的检测食品中PQ残留的间接ELISA法和GICA法,快速、方便,具有较好的实用价值,为ELISA检测试剂盒和金标试纸条检测食品中PQ的进一步研究提供了重要的实验依据。
Paraquat (PQ) is a bisquaternary ammonium compound used as a herbicide word-widely. There is a potential for human exposure to PQ in general population through residues on food materials and water. However actual methods, such as HPLC, GC and UV, are time consuming and need expensive instruments. Thus, development of a rapid and convenient detection method for local PQ analysis is extremely desirable. In this paper, enzyme-linked immunosorbent assay (ELISA) and gold-immunochromatography assay (GICA) involving the use of colloidal gold-labeled polyclonal antibody (PcAb) specific to PQ as the marker were developed for the analysis of PQ in foods.
First, N-(4-carboxybutyl)-N’-methylbipyridilium (PQ-h) was synthesized by using 4,4’-Bipyridine reacted with iodomethane by three steps. This experiment was conducted in dark under the protection of nitrogen and the products were identified through LC/MS, 1HNMR and IR, showing the purity and yield of the PQ-h were 96% and 69%, respectively. Then, immunogen (PQ-h-BSA) and coating antigen (PQ-h-OVA) were prepared according to the method of mixed anhydride. The ratio of PQ-hapten to BSA was 6:1.
Next, the PcAb to PQ-h-BSA was produced by immunizing New Zealand rabbits, and the titre of the antiserum was 51200. Then, an indirect ELISA for the determination of PQ was developed and the optimal working concentrations of the coating antigen and antisera were determined to be 0.3μg/mL and 1:5000 respectively after several chess board tests. The IC50 and LOD of this method were 5.13 ng/mL and 0.005 ng/mL, respectively, and the cross reactivity between PQ and didquat was less than 0.1%. Besides, the recoveries of PQ in soybean were ranged from 70% to 80% and the relative standard deviation (RSD) was within 10%.
Finally, colloidal gold was prepared through reducing HAuCl4·3H2O by sodium citrate and PcAb specific to PQ was conjugated to the gold nanoparticles under friendly and optimal condition as follows: optimum pH for conjugation was determined to be 8.5 and the least stable content of PcAb to colloidal gold was 8.1μg per milliliter gold solution. Then, a rapid, signal step and sensitive colloidal gold labeled immunochromatographic (GICA) test for detection of PQ in foods was developed and the optimum concentration of coating antigen and anti-rabbit IgG were 0.5 mg/ml and 0.11 mg/ml, respectively. This method is rapid, convenient and with high repeatability and stability, as the testing time is about 5 min, the lowest visual observation limit was 10 ng/ml and the shelf-life of immunostrips was at least five months at room temperature. Besides, the recoveries of PQ in soybean was good.
In conclusion, the ELISA and GICA developed to determine PQ residues in foods have good practical value and provide an important experimental basis for the further study on ELISA testing kits and immunochromatography testing paper.
首先以4,4’-联吡啶和碘甲烷为起始原料,经三步反应合成了百草枯半抗原N-甲基-N′-戊酸基-二吡啶二溴化物,经LC/MS、1HNMR和IR鉴定结果表明,所得产物即为百草枯半抗原(简称PQ-h),其纯度为96%,得率为69%。通过混合酸酐法偶联大分子蛋白载体牛血清白蛋白(BSA)和卵清蛋白(OVA)制备免疫抗原(PQ-h-BSA)和包被抗原(PQ-h-OVA),并进行结构鉴定。利用分光光度法测定百草枯半抗原与BSA的偶联比为6:1。
通过免疫新西兰大白兔,获得特异性多克隆抗体,效价为51200。建立了PQ检测的间接竞争ELISA方法,经过多次棋盘试验,最终确立最适包被抗原浓度为0.3μg/mL,抗体最佳稀释度为1:5000。该方法的半数抑制率IC50为5.13 ng/mL,最低检出限为0.005 ng/mL。百草枯与敌草快的交叉反应率小于0.1%。大豆样品的加标回收率在70%~80%之间,相对标准偏差(RSD)低于10%。
采用柠檬酸三钠还原法制备胶体金,通过研究胶体金与抗体蛋白作用过程,确定稳定标记1 ml胶体金需8.1μg抗体蛋白,标记体系的最佳pH为8.5。以金标抗体作为分析探针,PQ-h-OVA作为竞争抗原,以羊抗兔IgG为控制抗体,构建直接竞争胶体金标免疫层析检测体系。确定膜上包被抗原和羊抗兔二抗的最佳包被浓度分别为0.5 mg/ml和0.11 mg/ml。金标目测检出限为10 ng/ml,检测时间约5 min。方法的重复性和稳定性较好,经推测,该试纸条在室温下至少可以保存5个月。对大豆进行加标回收试验,回收率较好。
本研究所建立的检测食品中PQ残留的间接ELISA法和GICA法,快速、方便,具有较好的实用价值,为ELISA检测试剂盒和金标试纸条检测食品中PQ的进一步研究提供了重要的实验依据。
Paraquat (PQ) is a bisquaternary ammonium compound used as a herbicide word-widely. There is a potential for human exposure to PQ in general population through residues on food materials and water. However actual methods, such as HPLC, GC and UV, are time consuming and need expensive instruments. Thus, development of a rapid and convenient detection method for local PQ analysis is extremely desirable. In this paper, enzyme-linked immunosorbent assay (ELISA) and gold-immunochromatography assay (GICA) involving the use of colloidal gold-labeled polyclonal antibody (PcAb) specific to PQ as the marker were developed for the analysis of PQ in foods.
First, N-(4-carboxybutyl)-N’-methylbipyridilium (PQ-h) was synthesized by using 4,4’-Bipyridine reacted with iodomethane by three steps. This experiment was conducted in dark under the protection of nitrogen and the products were identified through LC/MS, 1HNMR and IR, showing the purity and yield of the PQ-h were 96% and 69%, respectively. Then, immunogen (PQ-h-BSA) and coating antigen (PQ-h-OVA) were prepared according to the method of mixed anhydride. The ratio of PQ-hapten to BSA was 6:1.
Next, the PcAb to PQ-h-BSA was produced by immunizing New Zealand rabbits, and the titre of the antiserum was 51200. Then, an indirect ELISA for the determination of PQ was developed and the optimal working concentrations of the coating antigen and antisera were determined to be 0.3μg/mL and 1:5000 respectively after several chess board tests. The IC50 and LOD of this method were 5.13 ng/mL and 0.005 ng/mL, respectively, and the cross reactivity between PQ and didquat was less than 0.1%. Besides, the recoveries of PQ in soybean were ranged from 70% to 80% and the relative standard deviation (RSD) was within 10%.
Finally, colloidal gold was prepared through reducing HAuCl4·3H2O by sodium citrate and PcAb specific to PQ was conjugated to the gold nanoparticles under friendly and optimal condition as follows: optimum pH for conjugation was determined to be 8.5 and the least stable content of PcAb to colloidal gold was 8.1μg per milliliter gold solution. Then, a rapid, signal step and sensitive colloidal gold labeled immunochromatographic (GICA) test for detection of PQ in foods was developed and the optimum concentration of coating antigen and anti-rabbit IgG were 0.5 mg/ml and 0.11 mg/ml, respectively. This method is rapid, convenient and with high repeatability and stability, as the testing time is about 5 min, the lowest visual observation limit was 10 ng/ml and the shelf-life of immunostrips was at least five months at room temperature. Besides, the recoveries of PQ in soybean was good.
In conclusion, the ELISA and GICA developed to determine PQ residues in foods have good practical value and provide an important experimental basis for the further study on ELISA testing kits and immunochromatography testing paper.
引文
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[2]吴志凤,秦冬梅.百草枯在土壤中的安全性及残留检测技术简介[J].农药科学与管理,2003,24(3):17-18
[3]宁宗,李其斌.百草枯中毒所致的肺损害及治疗进展[J].蛇志,2007,19(3):208-210
[4] Wagner,S.L.Clinical Toxicology of Agriculture Chemicals.Oregon State University Environmental Health Sciences Center,Corvallis,OR,1981.10-33
[5]庄英男.除草剂百草枯的毒理安全性研究[D]:[硕士学位论文].长春:吉林农业大学,2007
[6]夏敏,刘建华.百草枯中毒研究现状[J].四川医学,1995,16(4):237-239
[7]冯亚宾,崔朝勃.百草枯中毒的肺部损害[J].临床肺科杂志,2006,11(5):638
[8]丁正同,任惠民,蒋雨平等.百草枯对新生鼠黑质纹体状体系统的影响[J].复旦学报(医学科学版),2001,28:28-31
[9] Tanner,CM.Goldman,SM.Epidemiology of Parkinson’s disease [J].Neuroepedemiology, 1996, 14:317-335
[10] Masafumi Tomita,Toshiko Okuyama,Hironobu Katsuyama,etc.Mouse model of paraquat-poisoned lungs and its gene expression profile[J].Toxicology, 2007, 231: 200-209
[11] Rioboo C,Franqueira D,Canle ML,et al.Microalgal bioassays as a test of pesticide photodegradation efficiency in water[J].Bull Environ Contam Toxicol, 2001,67: 233-238
[12] Khan SU.Determination of paraquat residues in food crops by gas chromatography[J].Bull Environ Contam Toxicol,1975,14(6):745-749
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