新烟碱类杀虫剂多标记时间分辨荧光免疫分析研究

Time-resolved fluorescence immunoassay (TRFIA) is a kind of detection technology of ultramicro quantitative immunoassay, which is better than radioimmunoassay and enzyme immunoassay. If the haptens or antiboies were labeled simultaneously by different lanthanide, pesticides muti-residue detection will be realized. Since the successful development of imidacloprid, neonicotinoid insecticide has become an important insecticides series. Several representative neonicotinoid insecticides were selected and the monoclonal antibodies were prepared through the synthesis of different haptens. Then the optimum combination of antigen and antibody was screened by heterologous and homologous assay. On this basis, more than two lanthanide ions were labeled to monoclonal antibodies of different neonicotinoid insecticides,respectively. using the difference between lanthanide ions on wavelength and attenuation time, the multi-labeled time-resolved fluorescence immunoassays were developed for simultaneous detection of more than two neonicotinoid insecticides through optimization of markers and enhancement solution. And it will be applied to detect the pesticides residue in agricultural products and environment. This will provide one new idea for development of immunoassay on pesticides multi-residue, and there is important significance to further development of pesticides multi-residue detection kit with independent intellectual property rights.

时间分辨荧光免疫分析（TRFIA）是优于放射免疫分析和酶免疫分析的一种超微量定量免疫分析检测技术。选择不同的镧系元素标记抗原或抗体，可实现小分子化合物的同时检测。新烟碱类杀虫剂自吡虫啉成功开发以来，已成为一个重要的杀虫剂品种系列。本项目以几种代表性的新烟碱类杀虫剂为研究对象，通过设计合成不同结构的半抗原，制备相应的单克隆抗体，并利用同源、异源及特异性分析筛选最佳的抗原抗体组合。在此基础上，将2种以上镧系离子分别标记到不同新烟碱类杀虫剂的单克隆抗体上，利用镧系离子波长和衰减时间方面的差异性，建立一套两种以上新烟碱类杀虫剂同时检测的多标记时间分辨荧光免疫检测体系，并应用于农产品和环境中的残留分析。将为化学农药多残留免疫分析开创新的研究思路，对进一步开发具有自主知识产权的农药多残留检测试剂盒有着重要的现实意义。

时间分辨荧光免疫分析（TRFIA）是优于放射免疫分析和酶免疫分析的一种超微量定量免疫分析检测技术。选择不同的镧系元素标记抗原或抗体，可实现小分子化合物的同时检测。新烟碱类杀虫剂自吡虫啉成功开发以来，已成为一个重要的杀虫剂品种系列。本项目设计合成了2种新烟碱类杀虫剂半抗原，制备了特异性单克隆抗体。在此基础上，采用直接竞争和间接竞争模式，通过荧光标记、条件优化、交叉反应、添加回收及相关性验证等研究，分别建立了噻虫胺和烯唑醇间接竞争双标记时间分辨荧光免疫分析方法及吡虫啉和对硫磷直接竞争双标记时间分辨荧光免疫分析方法。. 在优化条件下，噻虫胺和烯唑醇的IC50分别为5.08和13.14µg/L，最低检测限分别为0.021和0.029µg/L；方法除与呋虫胺（9.4%）和烯效唑（4.28%）有交叉反应外，与其他类似化合物基本无交叉反应；6种基质样品噻虫胺的添加回收率为79.3-107.4%，相对标准偏差（RSD）为2.4-8.4%；烯唑醇的添加回收率为80.8-108.7%，相对标准偏差为2.3-9.5%，符合农药残留实验准则要求。用TRFIA对采集的样品进行检测，并通过GC进行验证。结果表明，dual-TRFIA和GC对实际样品中噻虫胺和烯唑醇的残留检测结果相关性良好。. 在优化条件下，对硫磷和吡虫啉的IC50分别为10.87和7.08µg/L，最低检测限分别为0.025和0.028µg/L；方法除与甲基对硫磷（42.4%）和氯噻啉（103.4%）有交叉反应外，与其他类似化合物均基本交叉反应；5种基质样品中吡虫啉的添加回收率在78.9-104.2%之间，RSD为2.4-11.6%；对硫磷的添加回收率为81.5-110.9%，RSD为3.2-10.5%。用TRFIA对实际样品进行检测，并通过GC进行验证，结果表明，dual-TRFIA和GC对实际样品中对硫磷和吡虫啉的残留检测结果具有高度相关性。对硫磷的相关性方程为y=1.0251x+0.2936，决定系数R2为0.9918；吡虫啉的相关性方程为y=0.9848 x+1.61，决定系数R2为0.9908。进一步验证了dual-TRFIA的准确性和可靠性。.本项目研究拓宽了TRFIA的检测范围，为该技术在农药多残留检测领域的应用奠定了理论和实践基础，对进一步开发具有自主知识产权的农药多残留检测试剂盒有着重要的现实意义。