噻虫嗪抗独特型纳米抗体提高免疫分析灵敏度的分子机制研究

Immunological detection technology is one of the most important screening methods for pesticide residue. As a substitute for antigen, anti-idiotypic antibody can significantly improve the sensitivity of immunoassay, while, at present, the molecular mechanism of improving the analytical sensitivity of anti-idiotypic nanobody is unclear, which limits the application of anti-idiotypic antibody in the field of pesticide residues immunoassay. So the project intends to carry out the study on the molecular mechanism of thiamethoxam anti-idiotypic nanobody in improving the immunoassay sensitivity. In detail, ① producing anti-thiamethoxam monoclonal antibody. ② establishing phage display library, obtaining thiamethoxam phage display anti-idiotypic nanobody by affinity panning, preparation a series of anti-idiotypic nanobody with variable sensitivity by vitro evolution. ③ revealing the molecular mechanism of anti-idiotypic nanobody by molecular simulation. ④ exploration and verification of the relationship between the mechanism and sensitivity of anti-idiotypic nanobody, and verification of the mechanism. Project results can clarify study on the molecular mechanism of thiamethoxam anti-idiotypic nanobody in improving the immunoassay sensitivity, and provide a theoretical basis for the development and application of high-sensitivity anti-idiotypic nanobody.

免疫分析技术是农药残留现场快速筛查手段之一。抗独特型纳米抗体作为一种抗原替代物可显著提高免疫分析的灵敏度，然而农药抗独特型纳米抗体提高分析灵敏度的分子机制不明，成为限制抗独特型纳米抗体在农药残留免疫分析中应用的关键问题。本项目拟开展噻虫嗪抗独特型纳米抗体提高免疫分析灵敏度的分子机制研究，内容包括：①合成人工抗原，制备抗噻虫嗪单克隆抗体；②构建噬菌体展示纳米抗体文库，淘选噻虫嗪抗独特型纳米抗体，体外进化制备系列具有不同灵敏度的抗独特型纳米抗体；③分子模拟解析抗独特型纳米抗体与噻虫嗪抗体互作的分子机制；④比对分析探寻作用机制与分析灵敏度之间内在联系，并进行突变验证。研究结果有望阐明噻虫嗪抗独特型纳米抗体提高免疫分析灵敏度的分子机制，为其它农药高灵敏度的抗独特型纳米抗体的研制提供理论指导。

抗独特型纳米抗体作为一种抗原替代物可显著提高免疫分析的灵敏度，明确其提高分析灵敏度的分子机制，有利于抗独特型纳米抗体在农药残留免疫分析中的进一步应用。本项目利用噻虫嗪原药一步合成噻虫嗪半抗原，通过活泼酯法合成噻虫嗪人工抗原，筛选获得1株稳定分泌抗噻虫嗪单克隆抗体的杂交瘤细胞株。在最优条件下，建立了灵敏的噻虫嗪残留免疫分析方法，ELISA分析线性范围为0.125-25.0 ng/mL，IC50值2.11 ng/mL，最低检测限为0.17 ppb (IC10)。试纸条分析可视检测限为0.5 μg/L，2.0 μg/L完全消线，建立的免疫分析方法成功用于农产品中噻虫嗪残留快速筛查。构建了3种动物源的天然抗独特型纳米抗体噬菌体展示文库，文库容量约为2×1012，开展了噻虫嗪噬菌体展示抗独特型纳米抗体的淘选，有效富集到抗独特型纳米抗体噬菌体阳性克隆。本研究结果为深入探讨噻虫嗪抗独特型纳米抗体提高免疫分析灵敏度的分子机制提供了基础数据。