有机磷农药多残留酶联免疫吸附分析方法的研究
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摘要
本研究以建立环境中有机磷农药多残留的酶联免疫吸附分析(ELISA)方法为目标,设计合成了有机磷农药通用半抗原,通过结合载体蛋白质制备了通用人工抗原,免疫动物获得了抗多种有机磷农药的群特异性抗血清。在所获得抗体的基础上,建立了有机磷农药多残留ELISA方法。所建立的方法为有机磷农药的快速多残留检测提供了技术手段,具有一定的使用价值和推广应用前景。主要研究结果如下:
1.通过O,O-二乙基硫代磷酰氯和4-氨基丁酸反应合成了有机磷农药通用半抗原二乙基-N-(3-羧基丙基)硫逐磷酰胺酯(简称DENP),并进行了结构鉴定。
2.采用碳二亚胺法将DENP与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)进行偶联,制备了通用人工抗原DENP-BSA和包被原DENP-OVA。经鉴定,结合比分别为12︰1和17:1。
3.将通用人工抗原DENP-BSA免疫两只新西兰大白兔,采血获得了针对有机磷农药的群特异性抗血清,效价分别1:102400和1:25600。
4.考察了9种有机磷农药与抗血清的亲和性,发现与半抗原DENP结构高度相似的甲拌磷、乙拌磷,内吸磷与抗体的亲和性最好;与DENP同样含有直链烃结构,但另一端为二甲氧基(硫代)磷酸酯的氧乐果和乐果与抗体的亲和性次之;与DENP一样含有二乙氧基硫代磷酸酯结构,但另一端连接基团为芳环或杂环的辛硫磷、二嗪农、喹硫磷及毒死蜱和抗体的亲和性较弱。
5.建立了用于检测甲拌磷、内吸磷的间接竞争ELISA方法的标准曲线,检测限分别为0.0037mg/L和0.033mg/L。对水样中添加的甲拌磷和内吸磷进行测定,得回收率范围为108.00%~112.00%。
The aim of the research is developing multi-residue enzyme linked immunosorbant assay (ELISA) for organophosphorus pesticides (OPs). Generic hapten for OPs was synthesized and conjugated with protein to produce generic immunogen which was then injected into rabbits to generate generic antiserum. With the antiserum, ELISA method for several OPs was then established and could be applied for multi-residue detection of OPs. Main results are listed below:
1. Generic Hapten O,O-diethyl-N-(3-carboxypropyl) phosphoramidothioate (named DENP) was synthesized from the reaction between O,O-diethylthiophosphoryl chloride and 4-aminobutyric acid.
2. Generic immungen (DENP-BSA) and coating antigen (DENP-OVA) were synthesized by conjugating generic hapten DENP with bovine serum albumin (BSA) and ovalbumin (OVA), respectively. Conjugation ratios were 12:1 and 17:1 respectively.
3. Generic antiserum was obtained by immunizing two rabbits with DENP-BSA. Titers of antisera were 1︰102400 and 25600.
4. Affinity between antiserum and 9 OPs was studied. The results showed that: OPs of nearly the same structure with hapten DENP, including phorate, demeton S and disulfoton, had highest affinity with antiserum; O,O-dimethyl phosphorothioate pesticides including omethoate and dimethoate were less affinitive with antiserum, though they also had linear hydrocarbon structure like DENP; OPs contain aromatic or heterocyclic ring including phoxim, quinalphos, diazinon and chlorpyrifos had least affinity with antiserum, even they have the same O,O-diethyl phosphorothioate with DENP.
5. Multi-residue ELISA for phorate and demeton S was developed,with the LOD of 0.0037 mg/L and 0.033mg/L, respectively. And the recoveries of phorate and demeton S in the water samples were from 108.0% to 112.0%.
1.通过O,O-二乙基硫代磷酰氯和4-氨基丁酸反应合成了有机磷农药通用半抗原二乙基-N-(3-羧基丙基)硫逐磷酰胺酯(简称DENP),并进行了结构鉴定。
2.采用碳二亚胺法将DENP与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)进行偶联,制备了通用人工抗原DENP-BSA和包被原DENP-OVA。经鉴定,结合比分别为12︰1和17:1。
3.将通用人工抗原DENP-BSA免疫两只新西兰大白兔,采血获得了针对有机磷农药的群特异性抗血清,效价分别1:102400和1:25600。
4.考察了9种有机磷农药与抗血清的亲和性,发现与半抗原DENP结构高度相似的甲拌磷、乙拌磷,内吸磷与抗体的亲和性最好;与DENP同样含有直链烃结构,但另一端为二甲氧基(硫代)磷酸酯的氧乐果和乐果与抗体的亲和性次之;与DENP一样含有二乙氧基硫代磷酸酯结构,但另一端连接基团为芳环或杂环的辛硫磷、二嗪农、喹硫磷及毒死蜱和抗体的亲和性较弱。
5.建立了用于检测甲拌磷、内吸磷的间接竞争ELISA方法的标准曲线,检测限分别为0.0037mg/L和0.033mg/L。对水样中添加的甲拌磷和内吸磷进行测定,得回收率范围为108.00%~112.00%。
The aim of the research is developing multi-residue enzyme linked immunosorbant assay (ELISA) for organophosphorus pesticides (OPs). Generic hapten for OPs was synthesized and conjugated with protein to produce generic immunogen which was then injected into rabbits to generate generic antiserum. With the antiserum, ELISA method for several OPs was then established and could be applied for multi-residue detection of OPs. Main results are listed below:
1. Generic Hapten O,O-diethyl-N-(3-carboxypropyl) phosphoramidothioate (named DENP) was synthesized from the reaction between O,O-diethylthiophosphoryl chloride and 4-aminobutyric acid.
2. Generic immungen (DENP-BSA) and coating antigen (DENP-OVA) were synthesized by conjugating generic hapten DENP with bovine serum albumin (BSA) and ovalbumin (OVA), respectively. Conjugation ratios were 12:1 and 17:1 respectively.
3. Generic antiserum was obtained by immunizing two rabbits with DENP-BSA. Titers of antisera were 1︰102400 and 25600.
4. Affinity between antiserum and 9 OPs was studied. The results showed that: OPs of nearly the same structure with hapten DENP, including phorate, demeton S and disulfoton, had highest affinity with antiserum; O,O-dimethyl phosphorothioate pesticides including omethoate and dimethoate were less affinitive with antiserum, though they also had linear hydrocarbon structure like DENP; OPs contain aromatic or heterocyclic ring including phoxim, quinalphos, diazinon and chlorpyrifos had least affinity with antiserum, even they have the same O,O-diethyl phosphorothioate with DENP.
5. Multi-residue ELISA for phorate and demeton S was developed,with the LOD of 0.0037 mg/L and 0.033mg/L, respectively. And the recoveries of phorate and demeton S in the water samples were from 108.0% to 112.0%.
引文
1.关窝辉.有机磷农药对环境的污染.国外医学.卫生学分册. 1974 (1): 16-17.
2.杨军.我国食物中有机磷农药残留现状及干预措施.中国公共卫生. 2000 (8): 755-756.
3.刘萍,张进忠.有机磷农药残留检测技术研究进展.环境污染与防治. 2006 (1): 55-57.
4.周培,陆贻通.农药残留的酶联免疫检测技术研究进展.环境污染与防治, 2002(4): 248-251.
5.黎其万,潘灿平.农药残留免疫分析方法及其应用研究进展.西南农业学报, 2004, 17(2): 248-252.
6.焦奎,张书圣.酶联免疫分析技术及应用.北京:化学工业出版社. 2004: 208-220.
7.孙太凡,叶非.酶免疫分析技术在农药残留分析中的应用.理化检验-化学分册, 2005(41):223-226.
8. Vallejo R.P., Bogus E.R., Mumma R.O.. Effects of haptens structure and bridging groups on antisera specificity in parathion immunoassay development. J. Agric Food Chem. 1982, 30(3): 572-580.
9. Kim Y.J., Cho Y.A., Lee H.S., et al. Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity. Anal. Chim. Acta. 2003, 475 (1-2): 85-96.
10.董健,刘贤进,韩召军等.氟虫腈半抗原设计及抗体制备.江苏农业学报, 2001, 17(3): 172-175.
11.张存政,王冬兰,刘贤进等.对硫磷抗体制备及其性质分析.江苏农业学报, 2004, 20(1): 28-31.
12. Juan J.M., Jaime P., Angel M.. Development of enzyme-linked immunosorbent assays for the insecticide chlorpyrifos. 1 monoclonal antibody production and immunoassay design. J. Agric. Food Chem. 1996, 44: 4052-4062.
13.韩丽君,贾明宏,钱传范等.甲基对硫磷的酶联免疫吸附分析(ELISA)研究.农业环境科学学报, 2005, 24(1): 187-190.
14. Nanju L, McAdam D.P., Skerritt J.H.. Development of immunoassays for type II synthetic pyrethroids. 1. hapten design and application to heterologous and homologous assays. J. Agric. Food Chem. 1998, 46(2): 520-534.
15. Abad A., Moreno M.J., Montoya A.. Hapten synthesis and production of monoclonal antibodies to the N-methylcarbamate pesticide methiocarb. J. Agric. Food Chem. 1998, 46(6): 2417-2426.
16. Mercader J.V., Montoya A.. Development of monoclonal ELISAs for azinphos-methyl. 1. hapten synthesis and antibody production J. Agric. Food Chem. 1999, 47(3): 1276-1284.
17.张存政,刘贤进,余向阳等.对硫磷半抗原改造及其免疫抗体.南京农业大学学报, 2002, 25(4): 37-40.
18.刘媛,刘贤进,余向阳等. Milbemycin Oxime半抗原设计及其抗体制备.农药学学报, 2005, 7(3): 285-288.
19. Singh K.V., Jasdeep K, Varshney G.C., et al. Synthesis and characterization of hapten-protein conjugates for antibody production against small molecules. Bioconj. Chem. 2004, 15(1): 168-173.
20. Wong S. S. Chemistry of protein conjugation and cross-Linking. London: CRC Press: 101-127.
21.周培.克百威农药毒理及胶体金标记免疫测定技术研究.上海:上海交通大学.博士学位论文: 10-28.
22.唐建设,张敏,陆贻通.拟除虫菊酯类农药免疫分析人工抗原研究进展.科技通报. 2007, 23(6): 835-841.
23. Grabarek Z., Gergely J. Zero-length crosslinking procedure with the use of active esters. Anal. Biochem. 1990, 185(1): 131-135.
24. Hock B, Dankwardt, Kramer K, et al. Immunochemical techniques: antibody production for pesticide analysis: A review. Anal. Chim. Acta. 1995, 311(3): 393-405.
25. Marco M-P, Gee S, Hammock B D. Immunochemical techniques forenvironmental analysisⅡ. antibody production and immunoassay development. TrAC. 1995, 14(8): 415-425.
26. Tijssen P. Practice and theory of enzyme immunoassay. Amsterdam: Elsevier, 1985. 58-64.
27.张彦峰典型污染物涕灭威、多环芳烃和三丁基锡的酶联免疫吸附分析研究.天津:南开大学.博士学位论文: 9-39.
28. Hennion M-C, Barcelo D. Strengths and limitations of immunoassays for effective and efficient use for pesticide analysis in water samples: A review. Anal. Chim. Acta. 1998, 362(1): 3-34.
29. J. N. Banks, M. Q. Chaudhry, W. A. Matthews, et al. Production and characterisation of polyclonal antibodies to the common moiety of some organophosphorus pesticides and development of a generic type ELISA. Food and Agricultural Immunology. 1998, 10(4): 349-361.
30. J. C. Johnson, J. M. Van Emon, D. R. Pullman, et al. Development and evaluation of antisera for detection of the O,O-diethyl phosphorothionate and phosphorothionothiolate organophosphorus pesticides by immunoassay. Journal of Agricultural and Food Chemistry. 1998, 46(8): 3116-3123.
31. M. J. C. Alcocer, P. P. Dillon, B. M. Manning, et al. Use of phosphonic acid as a generic hapten in the production of broad specificity anti-organophosphate pesticide antibody. Journal of Agricultural and Food Chemistry. 2000, 48(6): 2228-2233.
32. M. S. Jang, S. J. Lee, X. Xue, et al. (2002). "Production and characterization of monoclonal antibodies to a generic hapten for class-specific determination of organophosphorus pesticides." Bulletin of the Korean Chemical Society. 2002, 23(8): 1116-1120.
33.李雪飞,杨艳刚,孙胜龙等.蔬菜中有机磷拟除虫菊酯类农药残留调查.环境与健康杂志. 2006,23(5):418-420.
34.朱国念,吴刚,吴慧明. 2003.有机磷杀虫剂毒死蜱人工抗原的合成与鉴定.中国农业科学. 2003,36(6):657-662.
35. Johnson J C, Van E J M, Pullman D R, Keeper K R. Development and evaluation of antisera for detection of the O,O-diethyl phosphorothionate andphosphorothionothiolate organophosphorus pesticides by immunoassay. J. Agric. Food Chem. 1998, 46(8): 3116-3123.
36.杨耀军,渠桂荣,孟凡涛,马光辉.西维因人工抗原的合成新方法.过程工程学报. 2005, 5(2): 201-204.
37. Chen J P S, Toribara T Y, Warner H. Microdetermination of phosphorus. Anal Chem. 1956, 28 (11): 1756-1758.
38. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analy. Biochem. 1976, 72 (1-2): 248-254.
39.袁玉璐,姚元. GB11893-89.水质总磷的测定-钼酸铵分光光度法.北京.国家环境保护局标准处. 1991.
40.郭鑫.动物免疫学实验教程.北京:中国农业大学出版社, 2007:11-17.
41.裘法祖编.现代免疫学实验技术.湖北:湖北科学技术出版社,1998:65-70
42. Kim Y J, Cho Y A, Lee H S, Lee Y T, Gee S J, Hammock B D. Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity. Anal Chim Acta. 2003(1-2), 475: 85-96.
43.谢晶曦.红外光谱在有机化学和药物化学中的应用.北京:北京大学出版社.1987:355-357.
44.石德时,周斌,覃雅丽等.氯霉素间接竞争ELISA(ciELISA)检测方法的建立.中国兽医学报. 2002, 22(1):77-79.
45. Hill A.S.; Skerritt J.H.; Bushway R.J.; et al. Development and application of laboratory and field immunoassays for chlorpyrifos in water and soil matrices. J. Agri. Food Chem. 1994, 42 (9): 2051-2058.
2.杨军.我国食物中有机磷农药残留现状及干预措施.中国公共卫生. 2000 (8): 755-756.
3.刘萍,张进忠.有机磷农药残留检测技术研究进展.环境污染与防治. 2006 (1): 55-57.
4.周培,陆贻通.农药残留的酶联免疫检测技术研究进展.环境污染与防治, 2002(4): 248-251.
5.黎其万,潘灿平.农药残留免疫分析方法及其应用研究进展.西南农业学报, 2004, 17(2): 248-252.
6.焦奎,张书圣.酶联免疫分析技术及应用.北京:化学工业出版社. 2004: 208-220.
7.孙太凡,叶非.酶免疫分析技术在农药残留分析中的应用.理化检验-化学分册, 2005(41):223-226.
8. Vallejo R.P., Bogus E.R., Mumma R.O.. Effects of haptens structure and bridging groups on antisera specificity in parathion immunoassay development. J. Agric Food Chem. 1982, 30(3): 572-580.
9. Kim Y.J., Cho Y.A., Lee H.S., et al. Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity. Anal. Chim. Acta. 2003, 475 (1-2): 85-96.
10.董健,刘贤进,韩召军等.氟虫腈半抗原设计及抗体制备.江苏农业学报, 2001, 17(3): 172-175.
11.张存政,王冬兰,刘贤进等.对硫磷抗体制备及其性质分析.江苏农业学报, 2004, 20(1): 28-31.
12. Juan J.M., Jaime P., Angel M.. Development of enzyme-linked immunosorbent assays for the insecticide chlorpyrifos. 1 monoclonal antibody production and immunoassay design. J. Agric. Food Chem. 1996, 44: 4052-4062.
13.韩丽君,贾明宏,钱传范等.甲基对硫磷的酶联免疫吸附分析(ELISA)研究.农业环境科学学报, 2005, 24(1): 187-190.
14. Nanju L, McAdam D.P., Skerritt J.H.. Development of immunoassays for type II synthetic pyrethroids. 1. hapten design and application to heterologous and homologous assays. J. Agric. Food Chem. 1998, 46(2): 520-534.
15. Abad A., Moreno M.J., Montoya A.. Hapten synthesis and production of monoclonal antibodies to the N-methylcarbamate pesticide methiocarb. J. Agric. Food Chem. 1998, 46(6): 2417-2426.
16. Mercader J.V., Montoya A.. Development of monoclonal ELISAs for azinphos-methyl. 1. hapten synthesis and antibody production J. Agric. Food Chem. 1999, 47(3): 1276-1284.
17.张存政,刘贤进,余向阳等.对硫磷半抗原改造及其免疫抗体.南京农业大学学报, 2002, 25(4): 37-40.
18.刘媛,刘贤进,余向阳等. Milbemycin Oxime半抗原设计及其抗体制备.农药学学报, 2005, 7(3): 285-288.
19. Singh K.V., Jasdeep K, Varshney G.C., et al. Synthesis and characterization of hapten-protein conjugates for antibody production against small molecules. Bioconj. Chem. 2004, 15(1): 168-173.
20. Wong S. S. Chemistry of protein conjugation and cross-Linking. London: CRC Press: 101-127.
21.周培.克百威农药毒理及胶体金标记免疫测定技术研究.上海:上海交通大学.博士学位论文: 10-28.
22.唐建设,张敏,陆贻通.拟除虫菊酯类农药免疫分析人工抗原研究进展.科技通报. 2007, 23(6): 835-841.
23. Grabarek Z., Gergely J. Zero-length crosslinking procedure with the use of active esters. Anal. Biochem. 1990, 185(1): 131-135.
24. Hock B, Dankwardt, Kramer K, et al. Immunochemical techniques: antibody production for pesticide analysis: A review. Anal. Chim. Acta. 1995, 311(3): 393-405.
25. Marco M-P, Gee S, Hammock B D. Immunochemical techniques forenvironmental analysisⅡ. antibody production and immunoassay development. TrAC. 1995, 14(8): 415-425.
26. Tijssen P. Practice and theory of enzyme immunoassay. Amsterdam: Elsevier, 1985. 58-64.
27.张彦峰典型污染物涕灭威、多环芳烃和三丁基锡的酶联免疫吸附分析研究.天津:南开大学.博士学位论文: 9-39.
28. Hennion M-C, Barcelo D. Strengths and limitations of immunoassays for effective and efficient use for pesticide analysis in water samples: A review. Anal. Chim. Acta. 1998, 362(1): 3-34.
29. J. N. Banks, M. Q. Chaudhry, W. A. Matthews, et al. Production and characterisation of polyclonal antibodies to the common moiety of some organophosphorus pesticides and development of a generic type ELISA. Food and Agricultural Immunology. 1998, 10(4): 349-361.
30. J. C. Johnson, J. M. Van Emon, D. R. Pullman, et al. Development and evaluation of antisera for detection of the O,O-diethyl phosphorothionate and phosphorothionothiolate organophosphorus pesticides by immunoassay. Journal of Agricultural and Food Chemistry. 1998, 46(8): 3116-3123.
31. M. J. C. Alcocer, P. P. Dillon, B. M. Manning, et al. Use of phosphonic acid as a generic hapten in the production of broad specificity anti-organophosphate pesticide antibody. Journal of Agricultural and Food Chemistry. 2000, 48(6): 2228-2233.
32. M. S. Jang, S. J. Lee, X. Xue, et al. (2002). "Production and characterization of monoclonal antibodies to a generic hapten for class-specific determination of organophosphorus pesticides." Bulletin of the Korean Chemical Society. 2002, 23(8): 1116-1120.
33.李雪飞,杨艳刚,孙胜龙等.蔬菜中有机磷拟除虫菊酯类农药残留调查.环境与健康杂志. 2006,23(5):418-420.
34.朱国念,吴刚,吴慧明. 2003.有机磷杀虫剂毒死蜱人工抗原的合成与鉴定.中国农业科学. 2003,36(6):657-662.
35. Johnson J C, Van E J M, Pullman D R, Keeper K R. Development and evaluation of antisera for detection of the O,O-diethyl phosphorothionate andphosphorothionothiolate organophosphorus pesticides by immunoassay. J. Agric. Food Chem. 1998, 46(8): 3116-3123.
36.杨耀军,渠桂荣,孟凡涛,马光辉.西维因人工抗原的合成新方法.过程工程学报. 2005, 5(2): 201-204.
37. Chen J P S, Toribara T Y, Warner H. Microdetermination of phosphorus. Anal Chem. 1956, 28 (11): 1756-1758.
38. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analy. Biochem. 1976, 72 (1-2): 248-254.
39.袁玉璐,姚元. GB11893-89.水质总磷的测定-钼酸铵分光光度法.北京.国家环境保护局标准处. 1991.
40.郭鑫.动物免疫学实验教程.北京:中国农业大学出版社, 2007:11-17.
41.裘法祖编.现代免疫学实验技术.湖北:湖北科学技术出版社,1998:65-70
42. Kim Y J, Cho Y A, Lee H S, Lee Y T, Gee S J, Hammock B D. Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity. Anal Chim Acta. 2003(1-2), 475: 85-96.
43.谢晶曦.红外光谱在有机化学和药物化学中的应用.北京:北京大学出版社.1987:355-357.
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