吡虫啉农药间接竞争ELISA检测方法的建立
|
摘要
为建立特异性检测吡虫啉的间接竞争酶联免疫分析方法(ic-ELISA),利用能特异性识别吡虫啉的单克隆抗体,对ic-ELISA步骤中相关参数进行优化,确定最佳反应条件。结果表明:建立的ic-ELISA检测方法的灵敏度(IC_(10))和检测范围(IC_(15)～IC_(85))分别为0.06、0.08～3.26μg·L~(-1)。该方法对大米、梨、卷心菜3种样品加标回收率为83.61%～112.65%,与商品化试剂盒检测结果相比,其决定系数(R~2)为0.953 1。这一研究建立的检测方法灵敏度高于大多数报道的检测方法,可满足我国规定的农产品中吡虫啉最大残留限量标准检测要求,用于谷物、果蔬中吡虫啉残留定量检测。
An indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) was developed for the detection of imidacloprid. The working conditions of ic-ELISA were optimized based on a specific monoclonal antibody against imidacloprid. The results showed that the sensitivity(IC_(10)) and the linear range(15%-85% inhibition) of the ic-ELISA were 0.06 and 0.08-3.26 μg·L~(-1) respectively. The rates of the recoveries from spiked rice, pear and cabbage were ranged from 83.61%-112.65%, these results showed a good correlation between the developed ELISA and commercial kit(R~2=0.953 1). The developed method is more sensitive than those in most publication and meets the maximum residue limits(MRLs) permitted by China, and can be used for quantitative detection of remaining imidacloprid in cereal, fruit and vegetable.
An indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) was developed for the detection of imidacloprid. The working conditions of ic-ELISA were optimized based on a specific monoclonal antibody against imidacloprid. The results showed that the sensitivity(IC_(10)) and the linear range(15%-85% inhibition) of the ic-ELISA were 0.06 and 0.08-3.26 μg·L~(-1) respectively. The rates of the recoveries from spiked rice, pear and cabbage were ranged from 83.61%-112.65%, these results showed a good correlation between the developed ELISA and commercial kit(R~2=0.953 1). The developed method is more sensitive than those in most publication and meets the maximum residue limits(MRLs) permitted by China, and can be used for quantitative detection of remaining imidacloprid in cereal, fruit and vegetable.
引文
[1] LEE J K,AHN K C,PARK O S,et al.Development of an ELISA for the detection of the residues of the insecticide imidacloprid in agricultural and environmental samples [J].J Agric Food Chem,2001,49(5):2159-2167.
[2] 郭婧怡,高宇,田英.吡虫啉暴露及其遗传毒性研究进展 [J].环境与职业医学,2017,34(11):1013-1018.
[3] LONARE M,KUMAR M,RAUT S,et al.Evaluation of imidacloprid-induced neurotoxicity in male rats:a protective effect of curcumin [J].Neurochem Int,2014,78:122-129.
[4] BAL R,NAZIROGLU M,TURK G,et al.Insecticide imidacloprid induces morphological and DNA damage through oxidative toxicity on the reproductive organs of developing male rats [J].Cell Biochem Funct,2012,30(6):492-499.
[5] KAPOOR U,SRIVASTAVA M K,SRIVASTAVA L P.Toxicological impact of technical imidacloprid on ovarian morphology,hormones and antioxidant enzymes in female rats [J].Food Chem Toxicol,2011,49(12):3086-3089.
[6] COSTA C,SILVARI V,MELCHINI A,et al.Genotoxicity of imidacloprid in relation to metabolic activation and composition of the commercial product [J].Mutat Res,2009,672(1):40-44.
[7] FENG S L,KONG Z M,WANG X M,et al.Assessing the genotoxicity of imidacloprid and RH-5849 in human peripheral blood lymphocytes in vitro with comet assay and cytogenetic tests [J].Ecotoxicol Environ Saf,2005,61(2):239-246.
[8] 王小青,练爱敏,李献.优化高效液相色谱法检测果蔬中吡虫啉残留 [J].广州化工,2018,46(10):93-95.
[9] 张武,李二虎,李桂红,等.超高效液相色谱串联质谱测定芹菜中味鲜胺、噻虫嗪等5种农药残留量 [J].农药科学与管理,2018,39(4):40-44.
[10] 李国烈,杜鑫,苏旭,等.超高效液相色谱-串联质谱方法测定桑葚中吡虫啉和啶虫脒残留量 [J].农药科学与管理,2018,39(1):41-46.
[11] 覃国新,劳水兵,莫仁甫,等.超高效液相色谱串联质谱法测定上海青基质中吡虫啉和嘧霉胺残留 [J].现代食品科技,2017,33(12):262-266.
[12] 诸力,王晨,陈红平,等.超高效液相色谱-串联质谱法同时测定茶叶中11种植物生长调节剂及吡虫啉、啶虫脒的残留 [J].分析化学,2017,45(4):529-536.
[13] 高玉玲,刘少彬,孙鹏.UHPLC-MS/MS法测定大米中丁草胺和吡虫啉残留 [J].黑龙江八一农垦大学学报,2018,30(1):33-37.
[14] 李广领,姜金庆,陈锡岭,等.吡虫啉残留酶联免疫吸附检测方法的建立 [J].食品科学,2011,32(12):228-233.
[15] 马寅生,冯才伟,贾芳芳,等.一种吡虫啉的酶联免疫快速检测试剂盒的研制 [J].山东畜牧兽医,2013,34(5):6-8.
[16] FANG Q K,WANG L M,CHENG Q,et al.A bare-eye based one-step signal amplified semiquantitative immunochromatographic assay for the detection of imidacloprid in Chinese cabbage samples [J].Analytica Chimica Acta,2015,881:82-89.
[17] 楼小华,高川川,朱文静,等.胶体金免疫层析法快速检测烟草中吡虫啉残留 [J].食品安全质量检测学报,2017,8(5):1739-1744.
[18] 刘运清,李岩松,乔斌,等.3种有机磷农药间接竞争ELISA检测方法的建立 [J].农药,2017,56(10):746-749.
[19] GB 2763-2016:食品中农药最大残留限量 [S].北京:中国标准出版社,2016.
[20] HUA X D,LIU X F,YIN W,et al.A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the detection of bifenthrin in a chemical soil barrier [J].Science of The Total Environment,2015,502:246-251.
[21] XU Z L,DENG H,DENG X F,et al.Monitoring of organophosphorus pesticides in vegetables using monoclonal antibody-based direct competitive ELISA followed by HPLC-MS/MS [J].Food Chemistry,2012,131(4):1569-1576.
[22] YIN W,HUA X D,LIU X F,et al.Development of an enzyme-linked immunosorbent assay for thiacloprid in soil and agro-products with phage-displayed peptide [J].Analytical Biochemistry,2015,481:27-32.
[23] KOERNER T B,ABBOTT M,GODEFROY S B,et al.Validation procedures for quantitative gluten ELISA methods:AOAC allergen community guidance and best practices [J].J AOAC Int,2013,96(5):1033-1040.
[24] 张姝.酶联免疫吸附测定技术在食品卫生检验中的应用 [J].生物技术世界,2015(6):69.
[25] 吴植,夏文龙,朱善元,等.鸭甲型肝炎病毒-Ⅰ型3D基因在昆虫细胞中的表达及抗原检测 [J].扬州大学学报(农业与生命科学版),2018,39(1):16-19.
[26] 张也,刘以祥.酶联免疫技术与食品安全快速检测 [J].食品科学,2003,24(8):200-204.
[27] 郑尊涛.杀虫剂吡虫啉和氟虫腈的酶联免疫吸附测定(ELISA)技术研究 [D].杭州:浙江大学,2004.
[28] 赵哲.胶体金法吡虫啉快速检测卡对蔬菜中吡虫啉农药残留快速检测的效果初探 [J].上海农业科技,2017(4):43.
[29] 姚蕾珺.吡虫啉、啶虫脒、克百威及涕灭威的免疫快速检测 [D].无锡:江南大学,2017.
[30] WANATABE S,ITO S,KAMATA Y,et al.Development of competitive enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies for chloronicotinoid insecticides imidacloprid and acetamiprid [J].Analytica Chimica Acta,2001,427(2):211-219.
[31] WANG R M,WANG Z H,YANG H,et al.Highly sensitive and specific detection of neonicotinoid insecticide imidacloprid in environmental and food samples by a polyclonal antibody-based enzyme-linked immunosorbent assay [J].Journal of the Science of Food and Agriculture,2012,92(6):1253-1260.
[32] 刘丽强,华朱鸣,许定花,等.卡那霉素酶联免疫检测方法的建立及检测条件的优化 [J].食品科学,2009,30(18):350-355.
[2] 郭婧怡,高宇,田英.吡虫啉暴露及其遗传毒性研究进展 [J].环境与职业医学,2017,34(11):1013-1018.
[3] LONARE M,KUMAR M,RAUT S,et al.Evaluation of imidacloprid-induced neurotoxicity in male rats:a protective effect of curcumin [J].Neurochem Int,2014,78:122-129.
[4] BAL R,NAZIROGLU M,TURK G,et al.Insecticide imidacloprid induces morphological and DNA damage through oxidative toxicity on the reproductive organs of developing male rats [J].Cell Biochem Funct,2012,30(6):492-499.
[5] KAPOOR U,SRIVASTAVA M K,SRIVASTAVA L P.Toxicological impact of technical imidacloprid on ovarian morphology,hormones and antioxidant enzymes in female rats [J].Food Chem Toxicol,2011,49(12):3086-3089.
[6] COSTA C,SILVARI V,MELCHINI A,et al.Genotoxicity of imidacloprid in relation to metabolic activation and composition of the commercial product [J].Mutat Res,2009,672(1):40-44.
[7] FENG S L,KONG Z M,WANG X M,et al.Assessing the genotoxicity of imidacloprid and RH-5849 in human peripheral blood lymphocytes in vitro with comet assay and cytogenetic tests [J].Ecotoxicol Environ Saf,2005,61(2):239-246.
[8] 王小青,练爱敏,李献.优化高效液相色谱法检测果蔬中吡虫啉残留 [J].广州化工,2018,46(10):93-95.
[9] 张武,李二虎,李桂红,等.超高效液相色谱串联质谱测定芹菜中味鲜胺、噻虫嗪等5种农药残留量 [J].农药科学与管理,2018,39(4):40-44.
[10] 李国烈,杜鑫,苏旭,等.超高效液相色谱-串联质谱方法测定桑葚中吡虫啉和啶虫脒残留量 [J].农药科学与管理,2018,39(1):41-46.
[11] 覃国新,劳水兵,莫仁甫,等.超高效液相色谱串联质谱法测定上海青基质中吡虫啉和嘧霉胺残留 [J].现代食品科技,2017,33(12):262-266.
[12] 诸力,王晨,陈红平,等.超高效液相色谱-串联质谱法同时测定茶叶中11种植物生长调节剂及吡虫啉、啶虫脒的残留 [J].分析化学,2017,45(4):529-536.
[13] 高玉玲,刘少彬,孙鹏.UHPLC-MS/MS法测定大米中丁草胺和吡虫啉残留 [J].黑龙江八一农垦大学学报,2018,30(1):33-37.
[14] 李广领,姜金庆,陈锡岭,等.吡虫啉残留酶联免疫吸附检测方法的建立 [J].食品科学,2011,32(12):228-233.
[15] 马寅生,冯才伟,贾芳芳,等.一种吡虫啉的酶联免疫快速检测试剂盒的研制 [J].山东畜牧兽医,2013,34(5):6-8.
[16] FANG Q K,WANG L M,CHENG Q,et al.A bare-eye based one-step signal amplified semiquantitative immunochromatographic assay for the detection of imidacloprid in Chinese cabbage samples [J].Analytica Chimica Acta,2015,881:82-89.
[17] 楼小华,高川川,朱文静,等.胶体金免疫层析法快速检测烟草中吡虫啉残留 [J].食品安全质量检测学报,2017,8(5):1739-1744.
[18] 刘运清,李岩松,乔斌,等.3种有机磷农药间接竞争ELISA检测方法的建立 [J].农药,2017,56(10):746-749.
[19] GB 2763-2016:食品中农药最大残留限量 [S].北京:中国标准出版社,2016.
[20] HUA X D,LIU X F,YIN W,et al.A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the detection of bifenthrin in a chemical soil barrier [J].Science of The Total Environment,2015,502:246-251.
[21] XU Z L,DENG H,DENG X F,et al.Monitoring of organophosphorus pesticides in vegetables using monoclonal antibody-based direct competitive ELISA followed by HPLC-MS/MS [J].Food Chemistry,2012,131(4):1569-1576.
[22] YIN W,HUA X D,LIU X F,et al.Development of an enzyme-linked immunosorbent assay for thiacloprid in soil and agro-products with phage-displayed peptide [J].Analytical Biochemistry,2015,481:27-32.
[23] KOERNER T B,ABBOTT M,GODEFROY S B,et al.Validation procedures for quantitative gluten ELISA methods:AOAC allergen community guidance and best practices [J].J AOAC Int,2013,96(5):1033-1040.
[24] 张姝.酶联免疫吸附测定技术在食品卫生检验中的应用 [J].生物技术世界,2015(6):69.
[25] 吴植,夏文龙,朱善元,等.鸭甲型肝炎病毒-Ⅰ型3D基因在昆虫细胞中的表达及抗原检测 [J].扬州大学学报(农业与生命科学版),2018,39(1):16-19.
[26] 张也,刘以祥.酶联免疫技术与食品安全快速检测 [J].食品科学,2003,24(8):200-204.
[27] 郑尊涛.杀虫剂吡虫啉和氟虫腈的酶联免疫吸附测定(ELISA)技术研究 [D].杭州:浙江大学,2004.
[28] 赵哲.胶体金法吡虫啉快速检测卡对蔬菜中吡虫啉农药残留快速检测的效果初探 [J].上海农业科技,2017(4):43.
[29] 姚蕾珺.吡虫啉、啶虫脒、克百威及涕灭威的免疫快速检测 [D].无锡:江南大学,2017.
[30] WANATABE S,ITO S,KAMATA Y,et al.Development of competitive enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies for chloronicotinoid insecticides imidacloprid and acetamiprid [J].Analytica Chimica Acta,2001,427(2):211-219.
[31] WANG R M,WANG Z H,YANG H,et al.Highly sensitive and specific detection of neonicotinoid insecticide imidacloprid in environmental and food samples by a polyclonal antibody-based enzyme-linked immunosorbent assay [J].Journal of the Science of Food and Agriculture,2012,92(6):1253-1260.
[32] 刘丽强,华朱鸣,许定花,等.卡那霉素酶联免疫检测方法的建立及检测条件的优化 [J].食品科学,2009,30(18):350-355.