除虫脲农药残留酶联免疫测定方法的研究
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摘要
除虫脲是一种理想的农作物杀虫剂,它杀虫范围大、药效高、用量少、毒性低、药效期长、对环境污染小、杀虫有选择,是一种很有前途的杀虫剂新品种。因此,对其在环境中残留问题的研究具有重要意义。为获得相关信息,所使用的检测方法必须具有对大量样本进行分析的能力。除虫脲测定的经典方法—气相色谱或高效液相色谱法虽然灵敏度高,但是费用昂贵且耗时耗力,因此需要一些改良的分析方法以便能在短时间内对大量样本进行低成本的分析。对于杀虫剂的分析来说,免疫检测法简单、迅速、精准、性价比高而且适用于实验室或室外,因此该方法已被证明是进行大量样本分析时的最佳选择。
自从1971年被首次应用以来,免疫分析法发展很快,目前已经成为一种比较成熟的杀虫剂残留物检测方法。本文主要研究了用直接竞争性酶联免疫吸附检测法(ELISA)检测土壤及水样中除虫脲含量的方法。文中合成了两种半抗原,分别用它们免疫动物制备抗体并合成酶标记物。经分析检测,选择出最适合的抗体并证明抗体对除虫脲的特异性良好。同时,文中还证明了所用免疫检测法灵敏度高、稳定性好,土壤(90%甲醇萃取)、水样及其它样品基质基本不对检测结果造成影响,对除虫脲的检测限可以达到0.05μg/dm~3。对土壤和水样的添加试验回收率均超过80%。
Diflubenzuron have desirable features and potential for use as insecticides, it has high insecticidal characteristic and low toxicity.And it has important value about how to analyse the residues in environment. In order to obtain this information,the capacity to analyse a large number of samples should be available. But the classical methods of diflubenzuron analysis by GC or HPLC are expensive and tedious, although they are of adequate sensitivity. Improved analytical methods are needed so that a large number of samples can be inexpensively analysed within a short period of time. Because immunoassay for pesticide analysis is simple, cost effective, and adaptable to laboratory or field, it has been proved to be the best choice for the analysis of the large number of samples.Since it was first used in 1971,immunochemical methods have been rapidly gained acceptance as analytical techniques for pesticide analysis. This paper described the development of sensitive competitive enzyme-linked immunosorbent assays (ELISAs) for quantitative detection of insecticide diflubenzuron in soil and water. Two haptens were synthesised for the production of diflubenzuron antibodies and used in the enzyme conjugate. After analysed, the best antibody was chosen and the developed assay was very specific to diflubenzuron. In the paper, a highly sensitive and specific ELISA method is described.No significant matrix effects from soil (90% methanol extraction), water and other pesticide residues were observed for the developed assay. The detection limit of this assay was 0.05 μ g/dm~3, and more than 80% recovery was obtained for diflubenzuron in soil and water recovery studies.
自从1971年被首次应用以来,免疫分析法发展很快,目前已经成为一种比较成熟的杀虫剂残留物检测方法。本文主要研究了用直接竞争性酶联免疫吸附检测法(ELISA)检测土壤及水样中除虫脲含量的方法。文中合成了两种半抗原,分别用它们免疫动物制备抗体并合成酶标记物。经分析检测,选择出最适合的抗体并证明抗体对除虫脲的特异性良好。同时,文中还证明了所用免疫检测法灵敏度高、稳定性好,土壤(90%甲醇萃取)、水样及其它样品基质基本不对检测结果造成影响,对除虫脲的检测限可以达到0.05μg/dm~3。对土壤和水样的添加试验回收率均超过80%。
Diflubenzuron have desirable features and potential for use as insecticides, it has high insecticidal characteristic and low toxicity.And it has important value about how to analyse the residues in environment. In order to obtain this information,the capacity to analyse a large number of samples should be available. But the classical methods of diflubenzuron analysis by GC or HPLC are expensive and tedious, although they are of adequate sensitivity. Improved analytical methods are needed so that a large number of samples can be inexpensively analysed within a short period of time. Because immunoassay for pesticide analysis is simple, cost effective, and adaptable to laboratory or field, it has been proved to be the best choice for the analysis of the large number of samples.Since it was first used in 1971,immunochemical methods have been rapidly gained acceptance as analytical techniques for pesticide analysis. This paper described the development of sensitive competitive enzyme-linked immunosorbent assays (ELISAs) for quantitative detection of insecticide diflubenzuron in soil and water. Two haptens were synthesised for the production of diflubenzuron antibodies and used in the enzyme conjugate. After analysed, the best antibody was chosen and the developed assay was very specific to diflubenzuron. In the paper, a highly sensitive and specific ELISA method is described.No significant matrix effects from soil (90% methanol extraction), water and other pesticide residues were observed for the developed assay. The detection limit of this assay was 0.05 μ g/dm~3, and more than 80% recovery was obtained for diflubenzuron in soil and water recovery studies.
引文
[1] 王小永,刁锡华.除虫脲的特性与合成[J].河北轻化工学院学报,1996(3),17:62～65
[2] 迟德富,严善春,张丹丹,姜兴林,王明玉.几种无公害药剂对球果花蝇的控制作用及其机理[J].东北林业大学学报,2002(9),30(5):58～62
[3] 陈小玉,姚武,李志远,陈琛.农药除虫脲的毒性实验[J].郑州大学学报(医学版),2002(3),2(37):202～203
[4] 张桥.卫生毒理学基础[M].北京:人民卫生出版社,2000:105
[5] 高晓辉等.除虫脲在苹果及土壤中的残留及消解动态的研究[J].农药,1989,28(1):7～8
[6] 宋长有.除虫脲生产的经济分析[J].现代化工,2000(7),20(7):48～51
[7] 肖平.新型高效低毒杀虫剂-除虫脲[J].农药,1975(6),27:37～41
[8] 张宗炳.新型杀虫剂的探索[J].江苏化工,1986(3):7～11
[9] 张甬元,徐盈,陈专.灭幼脲Ⅲ号与除虫脲在实验室鱼水系统中归趋的比较研究[J].水生生物学报,1991(9),15(3):207～210
[10] 华东化工学院分析化学教研组,成都科学技术大学分析化学教研组.分析化学[M].北京:高等教育出版社,1994:424
[11] 黄雅俊等.气相色谱法直接检测茶叶中除虫脲残留量[J].农药,1993(5),32:33
[12] 高斌富,金秀华,方赤光,方群欣,张永全,于微波.高效液相色谱法同时测定食品中除虫脲、灭幼脲和杀铃脲残留量[J].1996(7),24(7):865
[13] 天津轻工业学院,无锡轻工业学院,大连轻工业学院.工业发酵分析[M].北京:中国轻工业出版社,1997:95～140
[14] 栾丽君,贾华欣,刘云霞.除虫脲原药的高效液相色谱分析[J].农药,1995(7),34:20
[15] 黄永春,王继军,傅学起,黄士忠.SPE-HPLC测定黄瓜中除虫脲灭幼脲和杀铃脲的残留检测方法[J].农业环境科学学报,2004,23(3):615～618
[16] 胡颖圆,黄秀明,成秀娟.除虫脲、吡虫啉悬浮剂的液相色谱分析[J].农业科学与管理,2003,24(3):11～12
[17] 王功.大米除虫脲残留量测定[J].吉林粮食高等专科学校学报,2000(9),15(3):4～7
[18] Weir D. Handbook of experimental immunology [M]. Oxford: Blackwell Scientific Publications. 1990
[19] 邓安平,Franek Milan.酶联免疫吸附分析法测定水样中的阿特拉津[J].分析化学研究报告,1998(1),26(1):29～33
[20] 邓安平,杨红.酶联免疫吸附分析法测定土壤试样中的阿特拉津[J].分析化学研究简报,1999(6),27(6):657～660
[21] 汪美先等.免疫学基础[M].太原:山西科学技术出版社,1981:299～337
[22] 徐宜为等.免疫检测技术[M].北京:科学出版社,1991:158～192
[23] Tatake J G, Knapp M M, Ressler C. Synthesis and characterization of protein and polylysine conjugation of sulfamethoxazole and sulfanilic acid for investigation of sulfonamide drug[J]. Bioconjugate Chemistry, 1991, 2: 124～132
[24] Yu C C, Kuhr R J. Synthesis and insecticidal activity of substituted 1-phenyl-3- benzoylureas and 1-phenyl-3-benzoyl-2-thioureas[J]. Journal of Agricultural and Food Chemistry, 1976, 24: 134～136.
[25] 王浩丹,周申.生物医学标记示总踪技术[M].北京:人民卫生出版社,1995:99～130
[26] 朱忠勇等.实用医学检验学[M].北京:人民军医出版社,1992:790～792
[27] Ausubel, Frederick M, Brent. Short protocols in molecular biology[J]. John Wiley&Sons, Inc. 1998
[28] Wellings K, Mulder R, van Daalen J J. Synthesis and laboratory evaluation of 1-(2,6 disubstituted benzoyl)-3-phenylureas, a new class of insecticides[J]. Journal of Agricultural and Food Chemistry, 1973, 21:348～354
[29] Sheth H B, Sporns P. Development of a single ELISA for detection of sulfonamides[J]. Journal of Agricultural and Food Chemistry, 1991, 39:1696～1700
[30] Lommen A, Haasnoot W, Weseman J M. Nuclear magnetic resonance controlled method for coupling of fenoterol to a carrier and enzyme[J]. Food and Agricultural Immunology, 1995, 7: 123～129
[31] Paek S H, Bachas L G, Schramm W. Defined analyte-enzyme conjugates as signal generators in immunoassays[J]. Analytical Biochemistry, 1993, 210:145～154
[32] McAdam D P, Hill A S, Beasley H L, Skerritt J H. Mono-and polyclonal antibodies to the organophosphate fentrothion[J]. Journal of Agricultural and Food Chemistry, 1992, 40: 1466～1470
[33] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72: 248～254
[34] 杨廷彬,尹学念等.实用免疫学[M].长春:长春出版社,1994:365～381
[35] 卢锦汉,章以浩等.医学生物制品学[M].北京:人民卫生出版社,1995:121～139
[36] Akerstrom B, Brodin T, Reis K, Bjorck L. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies [J]. Journal of Immunology, 1995, 135: 2589～2592
[37] Burrin J, Newman D J. Production and assessment of antibodies, In Principles and Practice of Immunoassay[M]. New York: Stockton Press, 1991: 19～52
[38] Newsome W H, Yeung J M, Collins P G. Development of enzyme immunoassay for captan and its degradation product tetrahydrophthalimide in foods[J]. Journal of AOAC International, 1993, 76: 381～386
[39] Hill A S, McAdam D P, Edwards L, Skerritt JH. Quantitation of bioresmethrin, a synthetic pyrethroid grain protectant, by enzyme immunoassay[J]. Journal of Agricultural and Food Chemistry, 1993, 41: 2011～2018
[40] Averameas S, Ternynck T, Guesdon J L. Coupling of enzyme to antibodies and antigens[J]. Scandinavian Journal of Immunology Supplement, 1978, 8: 7～23
[41] Midgley A R, Nisender G D, Rebar R W. Principles for the assessment of reliability of radioimmunoassay methods (precision, accuracy, sensitivity, specificity) [J]. Acta Endocrinology, 1969, 63: 163～179
[42] Bull D L, Ivie G W. Fateof diflubenzuron in cotton, soil, and rotational crops[J]. Journal of Agricultural and Food Chemistry, 1978, 26: 515～520
[2] 迟德富,严善春,张丹丹,姜兴林,王明玉.几种无公害药剂对球果花蝇的控制作用及其机理[J].东北林业大学学报,2002(9),30(5):58～62
[3] 陈小玉,姚武,李志远,陈琛.农药除虫脲的毒性实验[J].郑州大学学报(医学版),2002(3),2(37):202～203
[4] 张桥.卫生毒理学基础[M].北京:人民卫生出版社,2000:105
[5] 高晓辉等.除虫脲在苹果及土壤中的残留及消解动态的研究[J].农药,1989,28(1):7～8
[6] 宋长有.除虫脲生产的经济分析[J].现代化工,2000(7),20(7):48～51
[7] 肖平.新型高效低毒杀虫剂-除虫脲[J].农药,1975(6),27:37～41
[8] 张宗炳.新型杀虫剂的探索[J].江苏化工,1986(3):7～11
[9] 张甬元,徐盈,陈专.灭幼脲Ⅲ号与除虫脲在实验室鱼水系统中归趋的比较研究[J].水生生物学报,1991(9),15(3):207～210
[10] 华东化工学院分析化学教研组,成都科学技术大学分析化学教研组.分析化学[M].北京:高等教育出版社,1994:424
[11] 黄雅俊等.气相色谱法直接检测茶叶中除虫脲残留量[J].农药,1993(5),32:33
[12] 高斌富,金秀华,方赤光,方群欣,张永全,于微波.高效液相色谱法同时测定食品中除虫脲、灭幼脲和杀铃脲残留量[J].1996(7),24(7):865
[13] 天津轻工业学院,无锡轻工业学院,大连轻工业学院.工业发酵分析[M].北京:中国轻工业出版社,1997:95～140
[14] 栾丽君,贾华欣,刘云霞.除虫脲原药的高效液相色谱分析[J].农药,1995(7),34:20
[15] 黄永春,王继军,傅学起,黄士忠.SPE-HPLC测定黄瓜中除虫脲灭幼脲和杀铃脲的残留检测方法[J].农业环境科学学报,2004,23(3):615～618
[16] 胡颖圆,黄秀明,成秀娟.除虫脲、吡虫啉悬浮剂的液相色谱分析[J].农业科学与管理,2003,24(3):11～12
[17] 王功.大米除虫脲残留量测定[J].吉林粮食高等专科学校学报,2000(9),15(3):4～7
[18] Weir D. Handbook of experimental immunology [M]. Oxford: Blackwell Scientific Publications. 1990
[19] 邓安平,Franek Milan.酶联免疫吸附分析法测定水样中的阿特拉津[J].分析化学研究报告,1998(1),26(1):29～33
[20] 邓安平,杨红.酶联免疫吸附分析法测定土壤试样中的阿特拉津[J].分析化学研究简报,1999(6),27(6):657～660
[21] 汪美先等.免疫学基础[M].太原:山西科学技术出版社,1981:299～337
[22] 徐宜为等.免疫检测技术[M].北京:科学出版社,1991:158～192
[23] Tatake J G, Knapp M M, Ressler C. Synthesis and characterization of protein and polylysine conjugation of sulfamethoxazole and sulfanilic acid for investigation of sulfonamide drug[J]. Bioconjugate Chemistry, 1991, 2: 124～132
[24] Yu C C, Kuhr R J. Synthesis and insecticidal activity of substituted 1-phenyl-3- benzoylureas and 1-phenyl-3-benzoyl-2-thioureas[J]. Journal of Agricultural and Food Chemistry, 1976, 24: 134～136.
[25] 王浩丹,周申.生物医学标记示总踪技术[M].北京:人民卫生出版社,1995:99～130
[26] 朱忠勇等.实用医学检验学[M].北京:人民军医出版社,1992:790～792
[27] Ausubel, Frederick M, Brent. Short protocols in molecular biology[J]. John Wiley&Sons, Inc. 1998
[28] Wellings K, Mulder R, van Daalen J J. Synthesis and laboratory evaluation of 1-(2,6 disubstituted benzoyl)-3-phenylureas, a new class of insecticides[J]. Journal of Agricultural and Food Chemistry, 1973, 21:348～354
[29] Sheth H B, Sporns P. Development of a single ELISA for detection of sulfonamides[J]. Journal of Agricultural and Food Chemistry, 1991, 39:1696～1700
[30] Lommen A, Haasnoot W, Weseman J M. Nuclear magnetic resonance controlled method for coupling of fenoterol to a carrier and enzyme[J]. Food and Agricultural Immunology, 1995, 7: 123～129
[31] Paek S H, Bachas L G, Schramm W. Defined analyte-enzyme conjugates as signal generators in immunoassays[J]. Analytical Biochemistry, 1993, 210:145～154
[32] McAdam D P, Hill A S, Beasley H L, Skerritt J H. Mono-and polyclonal antibodies to the organophosphate fentrothion[J]. Journal of Agricultural and Food Chemistry, 1992, 40: 1466～1470
[33] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72: 248～254
[34] 杨廷彬,尹学念等.实用免疫学[M].长春:长春出版社,1994:365～381
[35] 卢锦汉,章以浩等.医学生物制品学[M].北京:人民卫生出版社,1995:121～139
[36] Akerstrom B, Brodin T, Reis K, Bjorck L. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies [J]. Journal of Immunology, 1995, 135: 2589～2592
[37] Burrin J, Newman D J. Production and assessment of antibodies, In Principles and Practice of Immunoassay[M]. New York: Stockton Press, 1991: 19～52
[38] Newsome W H, Yeung J M, Collins P G. Development of enzyme immunoassay for captan and its degradation product tetrahydrophthalimide in foods[J]. Journal of AOAC International, 1993, 76: 381～386
[39] Hill A S, McAdam D P, Edwards L, Skerritt JH. Quantitation of bioresmethrin, a synthetic pyrethroid grain protectant, by enzyme immunoassay[J]. Journal of Agricultural and Food Chemistry, 1993, 41: 2011～2018
[40] Averameas S, Ternynck T, Guesdon J L. Coupling of enzyme to antibodies and antigens[J]. Scandinavian Journal of Immunology Supplement, 1978, 8: 7～23
[41] Midgley A R, Nisender G D, Rebar R W. Principles for the assessment of reliability of radioimmunoassay methods (precision, accuracy, sensitivity, specificity) [J]. Acta Endocrinology, 1969, 63: 163～179
[42] Bull D L, Ivie G W. Fateof diflubenzuron in cotton, soil, and rotational crops[J]. Journal of Agricultural and Food Chemistry, 1978, 26: 515～520