【作者】 金茂俊；

【导师】 朱国念；

【作者基本信息】 浙江大学 ， 植物保护， 2009， 博士

【摘要】 在课题组现有ELISA分析方法的研究基础上,进一步深化研究方法的相关影响因素,如基质效应、漂移现象及不同ELISA分析方法等。在ELISA三种包被模式研究中,结果显示包被抗体法和包被二抗法在分析方法的方法灵敏度和数据平行性方面较包被抗原法更有优势。为建立具更高灵敏度且可商品化开发的免疫分析方法,本文对时间分辨荧光分析方法（TRFIA）和化学发光酶免疫分析方法（CLEIA）进行了系统的研究。通过对TRFIA分析方法荧光增强液中螯合剂β-NTA浓度、TOPO浓度及pH值等条件的筛选,得到了对于Eu³⁺和Sm³⁺这两种稀土离子具有良好性能的荧光增强液。在此基础上,通过对包被温度和时间、包被缓冲液、封闭物质、反应缓冲液等条件的优化。在优化基础上,建立了克百威的TRFIA分析方法并进行了标样添加回收率。结果表明,所建立的克百威TRFIA分析方法的检测灵敏度为1.86 ng/mL,样品添加回收率试验表明建立的克百威TRFIA分析方法可用于样品中克百威残留检测。通过对CLEIA化学发光增敏液中缓冲体系、发光增强剂、鲁米诺浓度及氧化剂浓度的筛选,得到具有良好性能的发光增敏液。在此基础上,通过对化学发光板、包被温度和时间、包被缓冲液、反应缓冲液及有机溶剂与浓度等条件的优化,结果显示:Costar白色发光板的吸附效果较佳,克百威抗体选择用0.1M,pH7.2Tris-HCl液作为包被缓冲液并在并在37℃包被两小时效果为佳,封闭物质选用1%胰蛋白胨（Tyrtone）且反应缓冲液选择0.05 M,pH7.2Tris-HCl液可得到最好的反应灵敏度和最大发光计数,有机溶剂中甲醇对CLEIA影响最小,反应体系中最终含量2.5%为佳。在此基础上,建立了克百威的CLEIA分析方法,其检测限(IC₁₀)为0.362 ng/mL,检测灵敏度(IC₅₀)为4.244 ng/mL,并通过LC-MS/MS数据确证。结果表明克百威CLEIA分析方法在灵敏度方面明显优于ELISA分析方法,并在样品检测中得到很好的应用,对多种样品的检测限已达到相关国家及CAC等设定的克百威MRL值。在构建成功化学发光免疫分析系统基础上,进一步将CLEIA分析方法应用到三唑磷、毒死蜱和对硫磷三种有机磷农药残留分析技术中,建立的三唑磷、毒死蜱和对硫磷三种有机磷农药的CLEIA分析方法IC₅₀值分别为0.852、21.01和2.247 ng/mL,IC₁₀值分别为0.063、0.694和0.203 ng/mL。并分别与三种农药的ELISA分析方法的检测灵敏度和检测限进行比较,结果显示,CLEIA分析方法的检测限均有了较大的提高,特别是三唑磷的CLEIA分析方法,相比于ELISA分析方法其检测灵敏度和检测限提高了2个数量级,显示出CLEIA分析方法在农药残留检测方面应用的良好前景。更多还原

【Abstract】 On the basis of the existing research of the author’s laboratory about the ELISA method,in-depth study about the effect of various factors which including matrix effect,drift phenomenon and different ELISA coating formats to ELISA method was investigated.Comparison of different ELISA formats showed that antibody coated format and the second antibody coated format had better performance in sensitivity and variation coefficient of determination than antigen coated format.In order to establish the immuno-chemical method which had higher sensitivity and good application prospect,the systematic experiments about time-resolved fluorescence immunoassay（TRFIA） and chemiluminescence ELISA immunoassay（CLEIA） were carried out.A variety of conditions of fluorescence enhancement solution including the concentration ofβ-NTA,concentration of TOPO and pH value were optimized, subsequently,the fluorescence enhancement solution which had good enhancement activity to both Eu³⁺ and Sm³⁺ was obtained.Based on the results,TRFIA conditions were optimized.On the basis of the optimized conditions mentioned above,the carbofuran TRFIA method was generated and recoveries of standards was determined. The sensitivity for detection,the IC₅₀ value was 38.55 ng/ml at a practical working concentration range from 1.86 to 798.8 ng/ml,and the limit of detection for carbofuran was 1.86 ng/ml.The mean variation coefficient of standard determination was 25.30%.Continued,analysis of carbofuran in spiked sample was finished. Unfortunately,there was no advantage about the carbofuran TRFIA method to the carbofuran ELISA method.Therefore,the research on carbofuran CLEIA method was carried out.The chemiluminescence sensitizer was obtained after a variety of conditions were optimized.On the basis of it,CLEIA method conditions for earbofuran was optimized and the optimum conditions were given as followed:Costar^? white plate for chemiluminescence was coated with the antibody in 0.1M,pH7.2 Tris-HC1 and the plate was incubated at 37℃for 2 hours.Also,1%typtone as blocking substance and 0.05 M,pH7.2Tris-HCl buffer as reacting buffer could improve the method’s sensitivity and get the largest light-emitting count.And 2.5%methanol was suitable for the competitive reaction.On the basis of optimum conditions,the carbofuran CLEIA method was generated successfully.The sensitivity for detection,the IC₅₀ value was 4.244 ng/ml at a practical working concentration range from 0.362 to 49.76 ng/ml and the limit of detection for carbofuran was 0.362 ng/ml.Recoveries of carbofuran spiked into vegetable samples and environmental samples were determined by CLEIA after sample matrix effect testing.Finally,the confirmation test between CLEIA and LC-MS/MS was finished.The results showed that CLEIA method had higher sensitivity than ELISA method,and the limit of detection of a variety of samples reached the carbofuran’s MRL value laid down by CAC and relevant countries.On the success in generating the chemiluminescence immunoassay system,the analytical method was applied in determination of organophosphorus pesticides including triazophos,chlorpyrifos and parathion.The IC₅₀ value of CLEIA methods in determination of triazophos,chlorpyrifos and parathion were 0.852,21.01 and 2.247ng/mL,anad IC₁₀ value were 0.063,0.694 and 0.203ng/mL respectively. Subsequently,the results were compared with the results of ELISA methods.It showed that the LOD of CLEIA methods have been greatly improved,especially the triazophos CLEIA method.Compared to the ELISA method,the detection sensitivity and LOD were improved two orders of magnitude.It indicated that the CLEIA method had good prospects of application in detection of pesticide residue.更多还原