• 百种中国杰出学术期刊
  • 中国精品科技期刊
  • 中国高校百佳科技期刊
  • 中国高校精品科技期刊
  • 中国国际影响力优秀学术期刊
  • 中国科技核心期刊

留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

化学发光酶联免疫分析法同时检测3种有机磷农药残留

邹茹冰 柳颖 王双节 张亚 郭逸蓉 朱国念

邹茹冰, 柳颖, 王双节, 张亚, 郭逸蓉, 朱国念. 化学发光酶联免疫分析法同时检测3种有机磷农药残留[J]. 农药学学报, 2017, 19(1): 37-45. doi: 10.16801/j.issn.1008-7303.2017.0006
引用本文: 邹茹冰, 柳颖, 王双节, 张亚, 郭逸蓉, 朱国念. 化学发光酶联免疫分析法同时检测3种有机磷农药残留[J]. 农药学学报, 2017, 19(1): 37-45. doi: 10.16801/j.issn.1008-7303.2017.0006
Rubing ZOU, Ying LIU, Shuangjie WANG, Ya ZHANG, Yirong GUO, Guonian ZHU. Development and evaluation of chemiluminescence enzyme-linked immunoassay for residue detection of three organophosphorus pesticides[J]. Chinese Journal of Pesticide Science, 2017, 19(1): 37-45. doi: 10.16801/j.issn.1008-7303.2017.0006
Citation: Rubing ZOU, Ying LIU, Shuangjie WANG, Ya ZHANG, Yirong GUO, Guonian ZHU. Development and evaluation of chemiluminescence enzyme-linked immunoassay for residue detection of three organophosphorus pesticides[J]. Chinese Journal of Pesticide Science, 2017, 19(1): 37-45. doi: 10.16801/j.issn.1008-7303.2017.0006

化学发光酶联免疫分析法同时检测3种有机磷农药残留

doi: 10.16801/j.issn.1008-7303.2017.0006
基金项目: 

公益性行业(农业)科研专项 201203094-3

详细信息
    作者简介:

    邹茹冰, 女, 硕士研究生, E-mail:zourubing@zju.edu.cn

    通讯作者:

    郭逸蓉, 通信作者(Author for correspondence), 女, 博士, 讲师, 研究方向为农药残留分析, E-mail:yirongguo@zju.edu.cn

  • 中图分类号: S482.33;TQ450.263

Development and evaluation of chemiluminescence enzyme-linked immunoassay for residue detection of three organophosphorus pesticides

  • 摘要: 利用能同时识别对硫磷、甲基对硫磷和杀螟硫磷的宽谱特异性单克隆抗体,建立了同时测定这3种有机磷农药残留的化学发光酶联免疫分析方法(chemiluminescence enzyme immunoassay,CLEIA),比较了间接竞争(ic-CLEIA)和直接竞争(dc-CLEIA)2种反应模式,优化了相关理化参数,确立了最适反应条件。结果表明:间接竞争CLEIA法检测对硫磷、甲基对硫磷和杀螟硫磷的抑制中浓度(IC50)分别为5.57、2.30和2.62 μg/kg,检测线性范围分别为0.39~100、0.39~25和0.39~25 μg/kg;直接竞争CLEIA法检测对硫磷、甲基对硫磷和杀螟硫磷的抑制中浓度(IC50)分别为5.43、1.34和1.24 μg/kg,检测线性范围分别为0.39~100、0.10~25和0.10~25 μg/kg。所建立的CLEIA方法基本能满足对硫磷、甲基对硫磷和杀螟硫磷在谷物和果蔬中最大残留限量的检测要求,为研制有机磷农药多残留检测试剂盒提供了技术依据。
  • 图式 1  两种半抗原、对硫磷及类似物的化学结构

    图式 1.  Chemical structures of the two haptens used in the immunoassay, parathion and the similar analytes

    图  1  PBS缓冲液中不同甲醇含量对CLEIA的影响

    Figure  1.  Effect of methanol on CLEIA performance

    图  2  不同pH值的PB缓冲液对CLEIA的影响

    Figure  2.  Effect of pH (PB) on CLEIA performance

    表  1  宽谱特异性抗体7B2对3种农药的交叉反应率

    Table  1.   Cross reactivity of the broad-specific antibody 7B2 toward the three pesticides

    农药Pesticide 7B2 (PA-QA1-7B2)
    IC20
    /(μg/kg)
    IC50
    /(μg/kg)
    交叉反应率CR/%
    对硫磷parathion 4.42 26.20 100.00
    甲基对硫磷parathion-methyl 1.73 11.90 220.20
    杀螟硫磷fenitrothion 0.72 7.30 358.90
    下载: 导出CSV

    表  2  反应缓冲液中离子强度对CLEIA的影响

    Table  2.   Effects of ionic strength in reaction buffers on CLEIA performances

    反应缓冲液Reaction buffer pH 离子强度IS/(mol/L) IC50/(μg/kg) IC20/(μg/kg) 最大发光值RLUmax 最大发光值/IC50RLUmax/IC50
    ic-CLEIA 8.5 0.01 18.74 4.68 1.93 × 106 1.03 × 105
    0.05 11.91 2.67 1.75 × 106 1.47 × 105
    0.10 4.52 1.15 7.60 × 105 1.68 × 105
    dc-CLEIA 8.5 0.01 23.62 4.75 5.81 × 105 2.45 × 104
    0.05 18.09 3.83 5.98 × 105 3.31 × 104
    0.10 4.54 0.49 6.86 × 105 1.51 × 105
    下载: 导出CSV

    表  3  ic-CLEIA和dc-CLEIA方法理化参数比较

    Table  3.   Physical and chemical parameters of ic-CLEIA and dc-CLEIA

    理化参数Physicochemical parameters ic-CLEIA dc-CLEIA
    抗原用量Antigen 2 μg/mL 0.078 μg/mL
    抗体用量Antibody 0.312 5 μg/mL 2 μg/mL
    底物液体积Substrate volume 150 mL 120 mL
    有机溶剂Organic solvent 10%甲醇10% Methanol 10%甲醇10% Methanol
    反应缓冲液种类及pHThe type and pH of reaction buffer PB (8.5) PB (8.5)
    反应缓冲液离子强度Ionic strength of reaction buffer 0.10 mol/L 0.10 mol/L
    实验时间Reaction time 约2 hAbout 2 h 约1 hAbout 1 h
    线性范围及决定系数Linear range and coefficient of determination 对硫磷parathion 0.39~100 μg/kg (R2 > 0.98) 0.39~100 μg/kg (R2 > 0.99)
    甲基对硫磷parathion-methyl 0.39~25 μg/kg (R2 > 0.99) 0.10~25 μg/kg (R2 > 0.99)
    杀螟硫磷fenitrothion 0.39~25 μg/kg (R2 > 0.99) 0.10~25 μg/kg (R2 > 0.99)
    下载: 导出CSV

    表  4  CLEIA法对有机磷农药的IC50及交叉反应率

    Table  4.   IC50 and cross reactivity of CLEIA against organophosphorus pesticides

    农药Pesticide ic-CLEIA dc-CLEIA
    IC50/(μg/kg) 交叉反应率CR/% IC50/(μg/kg) 交叉反应率CR/%
    对硫磷parathion 5.57 100.00 5.43 100.00
    甲基对硫磷parathion-methyl 2.30 242.17 1.34 405.22
    杀螟硫磷fenitrothion 2.62 212.60 1.24 437.90
    倍硫磷fenthion > 1 000 < 1 > 1 000 < 1
    辛硫磷phoxim > 1 000 < 1 > 1 000 < 1
    水胺硫磷isocarbophos > 1 000 < 1 > 1 000 < 1
    毒死蜱chlorpyrifos > 1 000 < 1 > 1 000 < 1
    三唑磷triazophos > 1 000 < 1 > 1 000 < 1
    下载: 导出CSV

    表  5  有机磷农药多残留检测的ELISA、CLEIA分析方法比较

    Table  5.   ELISAs and CLEIAs for multi-residue detection of organophosphorus pesticide

    检测方法Detection methods 抗体类型Type of antibody 检测分析物的IC50/(μg/kg) IC50 of analytes/(μg/kg) 参考文献References
    对硫磷parathion 甲基对硫磷parathion-methyl 杀螟硫磷fenitrothion
    icELISA PAb > 1 000 483.90 324 [21]
    icELISA PAb NDa 447 1 023 [22]
    icELISA PAb 348 ND ND [23]
    icELISA MAb 64.20 78.40 43.90 [24]
    icELISA MAb 20.32 21.44 42.15 [25]
    icELISA MAb 1 430 2 150 ND [26]
    dcELISA MAb 1 × 105 2 × 106 ND [26]
    icELISA MAb 1.53 197.96 1 133.80 [27]
    icCLEIA PAb 1.14 7.05 321.43 [34]
    icCLEIA MAb 5.57 2.30 2.62
    dcCLEIA MAb 5.43 1.34 1.24
    ND: not detected.
    下载: 导出CSV

    表  6  采用dc-CLEIA方法添加3种有机磷农药的回收试验结果(n=3)

    Table  6.   Recovery test of three organophosphorus pesticides in samples (n=3)

    样品Sample 添加水平Spiked level/(μg/kg) 对硫磷parathion 甲基对硫磷parathion-methyl 杀螟硫磷fenitrothion
    检出量Found/(μg/kg) 回收率Recovery/% RSD/% 检出量Found/(μg/kg) 回收率Recovery/% RSD/% 检出量Found/(μg/kg) 回收率Recovery/% RSD /%
    苹果Apple 20 20.85 104 3.35 16.28 81 6.08 18.54 93 7.22
    100 101.63 102 9.45 91.04 91 8.97 94.13 94 9.80
    白菜Chinese cabbage 20 15.14 76 4.02 19.19 96 5.83 14.51 73 5.24
    100 96.05 96 9.92 100.66 101 1.70 86.72 87 6.12
    糙米Rice 40 42.47 106 6.40 34.55 86 11.18 34.92 87 10.12
    200 189.30 95 7.50 235.74 118 3.38 236.48 118 2.98
    下载: 导出CSV
  • 魏天龙.高效液相色谱法同时测定生活饮用水中七种有机磷农药残留研究[D].兰州:西北师范大学, 2013:5-9.

    WEI T L. The research of simultaneous determination of seven kinds of oragnicphosphorus pesticide residues in drinking water by high performance liquid chromatography[D]. Lanzhou:Northwest Normal University, 2013:5-9.
    BOUCHARD M F, CHEVRIER J, HARLEY K G, et al. Prenatal exposure to organophosphate pesticides and IQ in 7-year-old children[J]. Environ Health Perspect, 2011, 119(8):1189-1195. doi: 10.1289/ehp.1003185
    SHADNIA S, AZIZI E, HOSSEINI R, et al. Evaluation of oxidative stress and genotoxicity in organophosphorus insecticide formulators[J]. Hum Exp Toxicol, 2005, 24(9):439-445. doi: 10.1191/0960327105ht549oa
    REDMOND E, EGESTON C, BRALLEY J A. Low level prenatal exposure to organophosphate pesticides significantly lowers IQ in children[J]. Townsend Lett, 2012, 12(7):782-795. http://www.biomedsearch.com/article/Low-level-prenatal-exposure-to/276901133.html
    BANKS C N, LEIN P J. A review of experimental evidence linking neurotoxic organophosphorus compounds and inflammation[J]. Neurotoxicology, 2012, 33(3):575-584. doi: 10.1016/j.neuro.2012.02.002
    MONTUORI P, AURINO S, NARDONE A, et al. Spatial distribution and partitioning of organophosphates pesticide in water and sediment from Sarno River and Estuary, Southern Italy[J]. Environ Sci Pollut Res, 2015, 22(11):8629-8642. doi: 10.1007/s11356-014-4016-z
    四部委联合发布禁止高毒农药使用相关事宜的公告第632号[J].农药科学与管理, 2006, 25(7):1. http://mall.cnki.net/magazine/Article/NYKG200607000.htm

    The No. 632 of four ministries jointly issued a ban on high toxic pesticide use relevant matters announcement[J]. Pestic Sci Admin, 2006, 25(7):1. http://mall.cnki.net/magazine/Article/NYKG200607000.htm
    武丽辉.欧盟将更多农药列入淘汰使用名单[J].农药科学与管理, 2007, 28(11):15. http://www.cnki.com.cn/Article/CJFDTOTAL-NYKG200711009.htm

    WU L H. The European Union:more pesticide will be eliminated[J]. Pestic Sci Admin, 2007, 28(11):15. http://www.cnki.com.cn/Article/CJFDTOTAL-NYKG200711009.htm
    张宏军.澳大利亚禁用杀螟硫磷[J].农药科学与管理, 2004, 26(7):39. http://www.cnki.com.cn/Article/CJFDTOTAL-NYKG200407017.htm

    ZHANG H J. Fenitrothion was disabled in Australia[J]. Pestic Sci Admin, 2004, 26(7):39. http://www.cnki.com.cn/Article/CJFDTOTAL-NYKG200407017.htm
    HANDFORD C E, ELLIOTT C T, CAMPBELL K. A review of the global pesticide legislation and the scale of challenge in reaching the global harmonization of food safety standards[J]. Integr Environ Assess Manag, 2015, 11(4):525-536. doi: 10.1002/ieam.v11.4
    李培培, 陈敏, 王军. QuEChERS-高效液相色谱法检测红葡萄酒中多菌灵和甲霜灵杀菌剂残留[J].食品与发酵工业, 2015, 41(1):202-206. http://www.cnki.com.cn/Article/CJFDTOTAL-SPFX201501038.htm

    LI P P, CHEN M, WANG J. Determination of carbendazim and metalaxyl pesticides in red wine by QuEChERS-high performance liquid chromatography[J]. Food Ferment Indust, 2015, 41(1):202-206. http://www.cnki.com.cn/Article/CJFDTOTAL-SPFX201501038.htm
    ZHAO X S, KONG W J, WEI J H, et al. Gas chromatography with flame photometric detection of 31 organophosphorus pesticide residues in Alpinia oxyphylla dried fruits[J]. Food Chem, 2014, 162:270-276. doi: 10.1016/j.foodchem.2014.04.060
    WATANABE E, KOBARA Y, BABA K, et al. Aqueous acetonitrile extraction for pesticide residue analysis in agricultural products with HPLC-DAD[J]. Food Chem, 2014, 154:7-12. doi: 10.1016/j.foodchem.2013.12.075
    TAO X Q, CHEN M, JIANG H Y, et al. Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment[J]. Anal Bioanal Chem, 2013, 405(23):7477-7484. doi: 10.1007/s00216-013-7174-9
    肖勤, 林金明.化学发光免疫分析方法的应用研究进展[J].分析化学, 2015, 43(6):929-938. doi: 10.1016/S1872-2040(15)60831-3

    XIAO Q, LIN J M. Advances and applications of chemiluminescence immunoassay in clinical diagnosis and foods safety[J]. Chin J Anal Chem, 2015, 43(6):929-938. doi: 10.1016/S1872-2040(15)60831-3
    FEI Y, YU S C, YU L L, et al. Determination of residual enrofloxacin in food samples by a sensitive method of chemiluminescence enzyme immunoassay[J]. Food Chem, 2014, 149:71-75. doi: 10.1016/j.foodchem.2013.10.024
    任世奇, 王栩, 唐宝军, 等.微板式化学发光酶免疫分析法临床测定人血清中孕酮[J].分析化学, 2008, 36(6):729-734. doi: 10.1016/S1872-2040(08)60038-9

    REN S Q, WANG X, TANG B J, et al. Micro-plate chemiluminescence enzyme immunoassay for clinical determination of progesterone in human serum[J]. Chin J Anal Chem, 2008, 36(6):729-734. doi: 10.1016/S1872-2040(08)60038-9
    李源珍, 生威, 刘恩梅, 等.化学发光酶免疫法测牛奶中3种喹诺酮类药物[J].食品研究与开发, 2013, 34(16):78-81. http://www.cnki.com.cn/Article/CJFDTOTAL-SPYK201316024.htm

    LI Y Z, SHENG W, LIU E M, et al. Determination of QNs residues in milk by enhanced chemiluminescent enzyme immunoassay[J]. Food Res Dev, 2013, 34(16):78-81. http://www.cnki.com.cn/Article/CJFDTOTAL-SPYK201316024.htm
    MIKUIŠKA P, BRUŽEŇAK L, VEČEŘZ Z. Detection of peroxyacetyl nitrate in air using chemiluminescence aerosol detector[J]. Chem Papers, 2014, 68(1):1482-1490. https://www.researchgate.net/publication/265296839_Detection_of_peroxyacetyl_nitrate_in_air_using_chemiluminescence_aerosol_detector
    JANG M S, LEE S J, XUE X P, et al. Production and characterization of monoclonal antibodies to a generic hapten for-class-specific determination of organophosphorus pesticides[J]. Bull Korean Chem Soc, 2002, 23(8):1116-1119. doi: 10.5012/bkcs.2002.23.8.1116
    LIANG Y, LIU X J, LIU Y, et al. Synthesis of three haptens for the class-specific immunoassay of O, O-dimethyl organophosphorus pesticides and effect of hapten heterology on immunoassay sensitivity[J]. Anal Chim Acta, 2008, 615(2):174-183. doi: 10.1016/j.aca.2008.03.050
    LI Y L, ZHAO F C, ZHAO L Y, et al. Development of a broad-specificity immunoassay for determination of organophosphorus pesticides using dual-generic hapten antigens[J]. Food Anal Methods, 2015, 8(2):420-427. doi: 10.1007/s12161-014-9906-7
    XU Z L, XIE G M, LI Y X, et al. Production and characterization of a broad-specificity polyclonal antibody for O, O-diethyl organophosphorus pesticides and a quantitative structure-activity relationship study of antibody recognition[J]. Anal Chim Acta, 2009, 647(1):90-96. doi: 10.1016/j.aca.2009.05.025
    ZHAO F C, HU C Y, WANG H M, et al. Development of a MAb-based immunoassay for the simultaneous determination of O, O-diethyl and O, O-dimethyl organophosphorus pesticides in vegetable and fruit samples pretreated with QuEChERS[J]. Anal Bioanal Chem, 2015, 407(30):8959-8970. doi: 10.1007/s00216-015-9055-x
    王春梅.有机磷高特异性和宽谱单克隆抗体的筛选与比较研究[D].杭州:浙江大学, 2010:40-44.

    WANG C M. Preparation and comparison of high-specific and broad-selective monoclonal antibodies against organophosphorous pesticides[D]. Hangzhou:Zhejiang University, 2010:40-44.
    SHIM J Y, KIM Y A, LEE Y T, et al. Monoclonal antibody-based enzyme-linked immunosorbent assays for the organophosphorus insecticide O-ethylO-4-nitrophenyl phenylphosphonothioate (EPN)[J]. J Agric Food Chem, 2010, 58(9):5241-5247. doi: 10.1021/jf904528y
    XU Z L, ZENG D P, YANG J Y, et al. Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples[J]. J Environ Monit, 2011, 13(11):3040-3048. doi: 10.1039/c1em10331h
    KOLOSOVA A Y, PARK J H, EREMIN S A, et al. Comparative study of three immunoassays based on monoclonal antibodies for detection of the pesticide parathion-methyl in real samples[J]. Anal Chim Acta, 2004, 511(2):323-331. doi: 10.1016/j.aca.2004.01.047
    兰美静, 刘蕊, 郭逸蓉, 等.异源竞争ELISA法筛选有机磷农药宽谱单克隆抗体[C]//全国农药交流会论文集.上海:中国农药工业协会, 2014:225-230.

    LAN M J, LIU R, GUO Y R, et al. Heterologous screening of hybridomas for the development of broad-specific monoclonal antibodies against organophosphorus pesticides[C]//National Pesticide Conference. Shanghai:China Crop Protection Industry Association, 2014:225-230.
    食品中农药最大残留限量:GB2763-2014[S].北京:中国标准出版社, 2014. http://www.cnki.com.cn/Article/CJFDTotal-ZBZD201404008.htm

    Maximum residue limits for pesticides in food:GB2763-2014[S]. Beijing:Standards Press of China, 2014. http://www.cnki.com.cn/Article/CJFDTotal-ZBZD201404008.htm
    金茂俊.农药残留检测的荧光和化学发光免疫分析方法研究[D].杭州:浙江大学, 2009. http://www.cnki.com.cn/Article/CJFDTotal-FGGL201531058.htm

    JIN M J. Development of fluorescence and chemiluminescence immunoassays for the determination of pesticide residue[D]. Hangzhou:Zhejiang University, 2009. http://www.cnki.com.cn/Article/CJFDTotal-FGGL201531058.htm
    GUI W J, LIU Y H, WANG C M, et al. Development of a direct competitive enzyme-linked immunosorbent assay for parathion residue in food samples[J]. Anal Biochem, 2009, 393(1):88-94. doi: 10.1016/j.ab.2009.06.014
    ZENG K, YANG T B, ZHONG P, et al. Development of an indirect competitive immunoassay for parathion in vegetables[J]. Food Chem, 2007, 102(4):1076-1082. doi: 10.1016/j.foodchem.2006.06.050
    TANAKA R, TAKEMURA M, SATO M, et al. Comparison of chemiluminescence enzyme immunoassay (CLEIA) with ELISA for the determination of anti-cyclic citrullinated peptide antibodies.[J]. Int J Clin Chem, 2010, 411(1-2):22-25.
    邓浩, 孔德彬, 杨金易, 等.对硫磷化学发光酶联免疫吸附分析方法的建立和评价[J].分析化学, 2013, 41(2):247-252. http://www.cnki.com.cn/Article/CJFDTOTAL-FXHX201302014.htm

    DENG H, KONG D B, YANG J Y, et al. Development of an indirect competitive chemiluminescence enzyme-linked immunoassay for parathion[J]. Chin J Anal Chem, 2013, 41(2):247-252. http://www.cnki.com.cn/Article/CJFDTOTAL-FXHX201302014.htm
  • 加载中
图(3) / 表(6)
计量
  • 文章访问数:  1138
  • HTML全文浏览量:  159
  • PDF下载量:  112
  • 被引次数: 0
出版历程
  • 收稿日期:  2016-08-30
  • 录用日期:  2016-11-14
  • 刊出日期:  2017-02-01

目录

    /

    返回文章
    返回