基于纳米肽适配体的农药多残留高灵敏快速免疫分析方法的研究

Immunoassay is an important quick detection method for screening of multi-residues of pesticides. Due to their small molecular and single epitope, pesticides are usually detected by competitive immunoassay. However, the competitive immunoassays have several shortcoming including insufficient sensitivity, complicated detection procedure, which need separate bonded antibody-antigen from separated one. In this study, the molecular recognition of nanopaptamer was used as a new strategy to develop homogeneous noncompetitive immunoassay for multi-pesticide residues. Based on the broad-specificity monoclonal antibody of organophosphorus pesticides as described proviously, the immunocomplex is obtained by the generic-hapten of organophosphorus pesticides reacted with the monoclonal antibody. Second, the phage peptids that can recognize the immunocomplex are obtained using phage disply peptids library.Third, the phage peptids are used to set up nonopeptamers by molecular cloning and protein expression. And then, the monoclonal antiboy and nonopeptamer, which are labed with donor fluorophore and the acceptor fluorophore are used to developed TR-FRET homogeneously noncompetitive immunoassay for fast and high sensitive detection of multi-residus of organophosphorus pesticides. The data of this study will provide scientific basis for development of broad specific, high sensitive, rapid ELISA to multi- pesticide residues.

免疫检测技术是农药多残留快速检测的重要技术，由于农药分子量小、抗原表位单一，其免疫学分析方法目前多为竞争型，但这类方法存在着检测灵敏度不足；检测步骤繁琐需要将结合的抗体和抗原，与未结合的分离等问题。因此，本研究拟引入纳米肽适配体分子识别技术，结合均相非竞争免疫分析方法提出解决以上问题的新思路。本研究以前期研究获得的有机磷农药“宽谱特异性”单克隆抗体为基础；首先，将其与有机磷农药通用结构半抗原反应形成免疫复合物，然后，利用噬菌体展示肽库，筛选出能够识别免疫复合物的多肽，采用外源基因表达及分子改造技术，构建能够识别免疫复合物的纳米肽适配体，然后，用荧光供体标签和荧光受体标签分别标记单克隆抗体和纳米肽适配体，建立时间分辨荧光能量转移（TR-FRET）均相非竞争免疫分析技术，实现对农药多残留的高灵敏、快速检测。该研究将为建立灵敏、快速的农药多残留免疫检测技术提供科学依据。

免疫检测技术是农药多残留快速检测的重要技术，由于农药分子量小、抗原表位单一，其免疫学分析方法目前多为竞争型，但这类方法存在着检测灵敏度不足；检测步骤繁琐需要将结合的抗体和抗原，与未结合的分离等问题。因此，本研究拟引入纳米肽适配体分子识别技术，结合非竞争免疫分析方法提出解决以上问题的新思路。本研究利用噬菌体展示技术，从构建的p8展示的噬菌体随机环八肽库中筛选出了能够识别有机磷农药宽谱特异性单链抗体与有机磷农药形成的免疫复合物的噬菌体多肽；并利用外源基因表达及分子改造技术将其组装为性质稳定、生物安全的纳米肽适配体，并以此建立了非竞争免疫磁珠ELISA（ncMEISA）及TR-FRET均相非竞争免疫检测技术，用于有机磷农药多残留免疫分析，提高了有机磷农药多残留免疫分析的灵敏度，简化了检测步骤，缩短了检测时间，是农药多残留检测方法学的重要创新，拓展了分析方法的基础理论，具有重要科学意义。