拟除虫菊酯类农药的免疫和色谱分析方法研究

【作者】 胡礼鸣；

【导师】 杨亚玲；

【作者基本信息】 昆明理工大学 ， 化学工程， 2009， 硕士

【摘要】 拟除虫菊酯类农药是一类仿生杀虫剂,具有性质稳定,无特殊臭味及安全系数高,使用浓度低等众多优点,被广泛应用于农业、工业以及家具害虫的防治中。越来越多的动物实验表明：拟除虫菊酯可能会影响神经系统的发展,会诱发癌症,抑制免疫系统,还可能干扰内分泌系统。因此,拟除虫菊酯类农药在农作物、食品以及环境中的残留分析具有重要的意义。本论文以拟除虫菊酯类农药作为研究对象,研究该类农药的的免疫分析方法和化学仪器分析方法。本文综述了拟除虫菊酯类农药的检测分析方法的研究进展,从样品的前处理方法、化学仪器分析方法及免疫分析方法的三个方面进行总结；综述了免疫分析方法的性能特点及其在生物、药品、食品和环境等复杂样品中的检测应用；在此基础上确定了本论文的选题。实验部分参考Sally K. Mak等的方法,合成溴氰菊酯的半抗原及全抗原。利用全抗原免疫动物,并采用杂交瘤技术获得PRD的单克隆抗体。通过间接ELISA法、SDS-PAGE电泳法对纯化后的抗体的特异性进行考察。结果表明,抗体属于1gG1型；SDS-PAGE确定分子量为168 kDa;PRD的抗体与固定化抗原的亲和常数为4.75×108 L/mol;抗体的特异性良好。以获得的抗体为基础采用间接竞争ELISA法对拟除虫菊酯进行检测分析。优化反应条件,得到一条检测拟除虫菊酯类农药的工作曲线。选用超市购买的铁观音茶叶作为实验检测样品,用间接竞争ELISA方法测得加标回收率。确定了间接竞争ELISA方法在37℃下反应1.5 hr,溶液环境为pH7.6的PBS溶液为最佳反应条件。间接竞争ELISA方法测定PRD的最低检测限为89 pg/mL,回收率均在90%以上。研究将生物素-亲和素放大体系应用到ELISA当中,建立更为灵敏的拟除虫菊酯类农药分析测定方法。生物素-亲和素放大体系测定PRD的最低检测限为5 pg/mL。选用固相萃取法(SPE)对茶叶样品做前处理,与HPLC构建PRD-SPE-HPLC离线分析体系,对茶叶中的四种拟除虫菊酯类农药(和一种异构体)进行检测。对PRD-SPE-HPLC系统的分析使用条件进行了优化考察,最终选定甲醇为洗脱溶剂,70%乙腈水溶液为分析溶剂。采用PRD-SPE-HPLC系统检测茶叶中农药的加标回收率,四种农药的回收率稳定达到80%以上,一种农药异构体的回收率达到72.9%。从样品处理、结果灵敏度等方面将PRD-SPE-HPLC多目标分析系统与ELISA方法进行讨论。实验发现免疫分析方法具有高灵敏度,简便,快捷的特点,在拟除虫菊酯类农残的检测中很有优势。生物技术与化学方法相结合将是分析检测工作的趋势。更多还原

【Abstract】 Stable, free from unusual odor, safe, and low in concentration, pyrethroid is a type of bionic pesticide which is widely used in agriculture, industry as well as in the prevention and control of furniture pests. However, more and more animal experiments indicate that pyrethroid may impair neural development, induce cancer, suppress immunity system and interfere incretion system. Therefore, the determination of pyrethroid residue in crops, food and environment is of special significance. With pyrethroid as the analyte, this thesis studies the immunity analysis method and chemical and instrumental analysis methods of this pesticide.This paper gives an overview of the progresses achieved in studying the determination and analysis methods of pyrethroid pesticide, including the pre-treatment of samples, testing and analysis of chemical instrument, and immunity analysis. It then summarizes the properties and characteristics of immunity analysis and its application in the testing of biological, pharmaceutical, foodstuff, and environmental samples.In the part of experiment, deltamethrin hapten and antigen were synthesized by adopting the method recommended by Sally K. Mak et al. The experimental animal was immunized by antigen and a monoclonal antibody (McAb) specific for deltamethrin was produced by hybridoma technology. Then the chapter goes on to investigate the specificity of McAb by ELISA and SDS-PAGE. The results showed that PRD-McAb was of the 1gG1 type; that its molecular weight was 168 Kda.determined by SDS-PAGE; that the affinity constant of McAb with coated complete antigen was 4.75×108 L/mol; and that the antibody is differential.In the base of getting the McAb, we determined and analyzed the PRD by ELISA. After optimizing reaction condition, we obtained a calibration curve for free PRD by ELISA. Then, with Tie guan yin Tea bought from the supermarket as the sample, we detected the recoveries of PRD. The optimum condition of ELISA was determined at 37℃in temperature,1.5 hours in length, and pH7.6 in solution. The minimum detection limit was found to be 89 pg/mL by ELISA method and the recovery rate was above 90%. Furthermore, the biotin-avidin amplifying system was applied to establish a more sensitive determination method, the minimum detection limit of which was 5 pg/mL.We uesd’SPE in the pre-treatment of the tea sample, and established PRD-SPE-HPLC off-line analysis system which was then used in testing four kinds of pyrethroids. We optimized the condition of analysis in PRD-SPE-HPLC system, and chose methanol as the elution solvent, and 70% Acetonitrile/water as the analytical solvent. When tested by PRD-SPE-HPLC system, the recovery rate of four PRD pesticides in tea was above 80% while that of one pesticide isomer was 72.9%. Then this chapter makes a comparative study of the PRD-SPE-HPLC system and ELISA method in terms of sample treatment and sensitivity. We found that immunoassay method is high sensitive, simple and fast in determinating pyrethroid pesticide. Chapter Five summarizes the research process and anticipates what needs to be done in future researches. Combination of biotechnology and chemical methods will be a trend of analyse.更多还原