杀草丹半抗原及其人工抗原的制备与鉴定
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摘要
以乙基羟乙胺、升华硫、一氧化碳气体和对氯氯苄为原材料,合成羟基化杀草丹;经纯化后与琥珀酸酐反应,合成杀草丹半抗原,采用薄层层析色谱法、质谱法和红外光谱法分析鉴定为预期产物;利用碳二亚胺法将半抗原与牛血清蛋白BSA偶联制备杀草丹人工抗原,应用紫外光谱和荧光光谱法分析鉴定。结果表明,羟基化杀草丹经薄层层析展开效果良好,杀草丹半抗原经红外光谱分析具有明显的羧酸基团,有利于后续偶联操作;在26℃室温条件下制备的杀草丹人工抗原,其半抗原与载体蛋白偶联比为6.59∶1。杀草丹半抗原与杀草丹人工抗原的成功制备,可为进一步研制杀草丹抗体奠定基础。
2-( Ethylamino) ethanol,sublimed sulphur,carbon monoxide gas and p-chlorobenzyl chloride were used as raw materials to synthesize the hydroxyated thiobencarb through the chemical reaction. Then purified hydroxyated thiobencarb was reacted with succinic anhydride to synthesize the thiobencarb hapten,which was analyzed and identified by thin layer chromatography,mass spectrometry and infrared spectrum.The hapten was conjugated to bovine serum albumin BSA to prepare the artificial antigen of thiobencarb by the carbodiimide method,which was identified by ultraviolet spectrophotometry and fluorescence spectrophotometry. The results indicated that the effect of thin layer chromatography was well for hydroxyated thiobencarb.Infrared spectrum analysis showed that thiobencarb hapten had carboxylic acid perssad,which was facilitated subsequent coupling operations. And the coupling ratio of hapten and carrier protein was 6.59 ∶ 1 for artificial antigen of thiobencarb under the condition of room temperature was 26 ℃. The successful synthesis of hapten and artificial antigen of thiobencarb had deeply laid the foundation for the preparation of thiobencarb antibody.
2-( Ethylamino) ethanol,sublimed sulphur,carbon monoxide gas and p-chlorobenzyl chloride were used as raw materials to synthesize the hydroxyated thiobencarb through the chemical reaction. Then purified hydroxyated thiobencarb was reacted with succinic anhydride to synthesize the thiobencarb hapten,which was analyzed and identified by thin layer chromatography,mass spectrometry and infrared spectrum.The hapten was conjugated to bovine serum albumin BSA to prepare the artificial antigen of thiobencarb by the carbodiimide method,which was identified by ultraviolet spectrophotometry and fluorescence spectrophotometry. The results indicated that the effect of thin layer chromatography was well for hydroxyated thiobencarb.Infrared spectrum analysis showed that thiobencarb hapten had carboxylic acid perssad,which was facilitated subsequent coupling operations. And the coupling ratio of hapten and carrier protein was 6.59 ∶ 1 for artificial antigen of thiobencarb under the condition of room temperature was 26 ℃. The successful synthesis of hapten and artificial antigen of thiobencarb had deeply laid the foundation for the preparation of thiobencarb antibody.
引文
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[20]张绍娜,李子平,郭英香,等.呋喃唑酮人工抗原的制备[J].畜牧与饲料科学,2017,38(6):7-9.
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[22]李明.噻虫胺抗体制备及免疫分析方法研究[D].南京:南京农业大学,2015:36-37.
[23]赵建晖,林谷园,郑宇,等.超高效液相色谱-串联质谱法对鳗鱼及制品中禾草丹残留量的测定[J].福建分析测试,2009,18(1):38-41.
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[25]王宁.血清白蛋白与药物小分子的荧光光谱研究[D].长春:吉林大学,2015:25-35.
[26]罗舜菁,刘成梅,陈婷婷,等.对甲基苯甲酸连接的氯霉素全抗原合成与鉴定[J].食品工业科技,2011,32(3):200-203.
[27]雷红涛,庞杰,贺丽萍,等.半抗原-载体蛋白偶联物的三种电泳鉴定方法[J].中国农业科学,2010(3):565-570.
[28]孙秀兰,张银志,汤坚,等.黄曲霉毒素B1完全抗原构建中结合点位研究[J].食品与生物技术学报,2007,26(5):99-103.
[29]魏新林,刘莹,张少恩,等.氰戊菊酯特异性人工抗原的合成与鉴定[J].农产品加工,2009(3):5-9.
[30]齐小花,王正方,金泳,等.生物质谱法对人工抗原偶联比的快速测定[J].检验检疫科学,2007,17:Z56-Z57.
[31]孙凤霞,康立超,庞芳琴,等.克百威人工抗原合成及其免疫应用[J].西北农业学报,2018,27(1):131-137.
[32]桑光明.残杀威和氯氟氰菊酯的半抗原分子设计、合成及人工抗原制备[D].无锡:江南大学,2009:10.
[33]李明.噻虫胺抗体制备及免疫分析方法研究[D].南京:南京农业大学,2015:4.
[34]周思祥,刘福成.农药人工抗原的合成研究进展[J].农药,2005,44(8):337-340.
[2]Sabater C,Carrasco J M.Effects of thiobencarb on the growth of three species of phytoplankton[J].Bulletin of Environmental Contamination&Toxicology,1996,56(6):977-984.
[3]Khaled Y Abdel-Halim,Sanaa A Massoud.Oxidative stress and protein carbonylation induction in Mosquito Fish Gambusia affinis as biomarkers of Thiobencarb exposure[J].Sci Afric Publishers,2014,2(8):348-353.
[4]Fernández C.Thiobencarb-induced changes in acetylcholinesterase activity of the fish Anguilla anguilla[J].Pesticide Biochemistry and Physiology,2002,72(1):55-63.
[5]Sancho E,Fernández-Vega C,Ferrando M D,et al.Eel ATPase activity as biomarker of thiobencarb exposure[J].Ecotoxicology and Environmental Safety,2003,56(3):434-441.
[6]国家卫生计生委.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.
[7]常青云,周欣,高书涛,等.悬浮固化液相微萃取-气相色谱联用测定水样中酰胺类除草剂残留[J].分析化学,2012,40(4):523-528.
[8]章稳宏,张春兰,储西平.42%吡嘧·杀草丹可湿性粉剂的高效液相色谱分析[J].杂草学报,2011,29(4):37-39.
[9]吴昊,贺小敏,李爱民,等.ASE-GC/MS同时测定土壤中8种酰胺类除草剂[J].环境科学与技术,2018,41(2):122-127.
[10]沈金灿,谢丽琪,肖来龙,等.液相色谱-串联质谱法同时测定红茶和绿茶中7种硫代氨基甲酸酯类除草剂[J].分析化学,2009,37(12):1795-1799.
[11]Robert O Harrison,Marvin H Goodrow,Shirley J Gee,et al.Hapten synthesis for pesticide immunoassay development[J].Acs Symposium Series-american Chemical Society,1990,451(1):14-27.
[12]Shiro Miyake,Shigekazu Ito,Yuki Yamaguchi,et al.Immunochemical approach for assay of herbicide thiobencarb[J].Analytia Chimica Acta,1998,376(1):97-101.
[13]徐丽广.酞酸酯类药物残留分析方法的研究[D].无锡:江南大学,2009:3-4.
[14]王晶,陈秀金,王方雨,等.硫代磷酸酯类有机磷药物抗原设计及免疫分析[J].中兽医医药杂志,2016(1):71-77.
[15]陈秋英.几种除草剂的合成研究[D].沈阳:东北大学,2010:9-12.
[16]Maftouh,Moussaif1 A,Moutaouakki l A,et al.Synthesis of haptens for immunoassay of Chlorpyrifos-ethyl as organophosphorus pesticides[J].Journal of Materials and Environmental Sciences,2017,8(3):878-883.
[17]刘蓉蓉,刘进贤,刘媛,等.菌核净残留免疫分析半抗原的制备和鉴定[J].食品科学,2007,28(2):223-225.
[18]Lu P,Wang Y,Wang Y,et al.In vitro synthesis of a type Atrichothecenes complete antigen from T-2 toxin[J].Food and Agricultural Immunology,2016,27(6):870-885.
[19]帅丽芳,赵勇,银涛,等.西维因人工抗原的合成与鉴定[J].山西农业大学学报,2017,37(10):749-753.
[20]张绍娜,李子平,郭英香,等.呋喃唑酮人工抗原的制备[J].畜牧与饲料科学,2017,38(6):7-9.
[21]武德鹏.猪瘟病毒E2蛋白碳纳米管抗原的制备及其初步研究[D].洛阳:河南科技大学,2015:26-27.
[22]李明.噻虫胺抗体制备及免疫分析方法研究[D].南京:南京农业大学,2015:36-37.
[23]赵建晖,林谷园,郑宇,等.超高效液相色谱-串联质谱法对鳗鱼及制品中禾草丹残留量的测定[J].福建分析测试,2009,18(1):38-41.
[24]朱明华,胡坪.仪器分析[M].北京:高等教育出版社,2008:298-307.
[25]王宁.血清白蛋白与药物小分子的荧光光谱研究[D].长春:吉林大学,2015:25-35.
[26]罗舜菁,刘成梅,陈婷婷,等.对甲基苯甲酸连接的氯霉素全抗原合成与鉴定[J].食品工业科技,2011,32(3):200-203.
[27]雷红涛,庞杰,贺丽萍,等.半抗原-载体蛋白偶联物的三种电泳鉴定方法[J].中国农业科学,2010(3):565-570.
[28]孙秀兰,张银志,汤坚,等.黄曲霉毒素B1完全抗原构建中结合点位研究[J].食品与生物技术学报,2007,26(5):99-103.
[29]魏新林,刘莹,张少恩,等.氰戊菊酯特异性人工抗原的合成与鉴定[J].农产品加工,2009(3):5-9.
[30]齐小花,王正方,金泳,等.生物质谱法对人工抗原偶联比的快速测定[J].检验检疫科学,2007,17:Z56-Z57.
[31]孙凤霞,康立超,庞芳琴,等.克百威人工抗原合成及其免疫应用[J].西北农业学报,2018,27(1):131-137.
[32]桑光明.残杀威和氯氟氰菊酯的半抗原分子设计、合成及人工抗原制备[D].无锡:江南大学,2009:10.
[33]李明.噻虫胺抗体制备及免疫分析方法研究[D].南京:南京农业大学,2015:4.
[34]周思祥,刘福成.农药人工抗原的合成研究进展[J].农药,2005,44(8):337-340.