夹心式免疫反应模型下重链抗体与有机磷农药分子识别取向的研究

The model of sandwich immuno-reaction (sIR) has great advantages in sensitivity, accuracy and detection limits. However, it is difficult to establish sIR for detection of pesticides, because of the single epitope of pesticide molecule. Our present researches have shown that the organophosphorus pesticides would be recognized by antibody when the phospholipids group adsorbed by zirconium oxide. Thus, the sandwich immuno-reaction model for organophosphorus has been developed. However, the development of the sIR is restricted because of the recognition orientation of antibody and antigen under the model was not clear. In this project, we selected chlorpyrifos as an object for our studies: Linked arm structures will be derived on different sites of chlorpyrifos compound by chemical method, and 5 kinds of haptens / immunogens to chlorpyrifos were synthesized. Immunogens were used to immunize the alpaca, the phage display heavy chain antibody library was established and the phage display heavy chain antibodies which can identify the adsorbed chlorpyrifos were obtained. The exposure direction of epitope of captured chlorpyrifos and relationship between the structure of haptens and the recognition orientation of heavy chain antibody was studied based on the combination of different recognition effects of heavy chain antibody to captured chlorpyrifos and the conformation、electronic distribution and the exposure direction of epitope of haptens. The stable binding form of heavy chain antibody and captured chlorpyrifos were established by using the molecular modeling software. Finally, the recognition orientation of heavy-chain antibody and target molecules adsorbed by zirconium oxide was revealed. Furthermore, this project would be to provide scientific guidance for the hapten design for establishment of sandwich immuno-reaction model for detection of organophosphorus pesticides.

夹心式免疫反应在灵敏度、准确度和检测范围等方面有较大优势，但农药小分子抗原表位单一，难以建立夹心式免疫反应模型。申请人前期研究证明：有机磷农药的磷氧基团能被氧化锆吸附，且吸附后能被抗体识别，形成夹心式免疫反应模型，但该模型下抗原抗体识别取向规律不明确，限制了该模型的发展。本项目拟以毒死蜱为研究对象：从毒死蜱分子不同方位引出连接臂，合成5种半抗原和人工抗原；免疫羊驼后，筛选针对不同半抗原的噬菌体展示重链抗体。比较不同半抗原相应的重链抗体与被捕获毒死蜱的识别效果，结合半抗原立体构象、电子分布以及抗原表位暴露方向等信息，研究被氧化锆捕获毒死蜱分子抗原表位的暴露形式以及半抗原结构与重链抗体识别定位的关系;利用计算机分子模拟手段建立重链抗体与目标分子结合的稳定空间形式，最终揭示重链抗体与被氧化锆吸附目标分子识别取向规律，为有机磷农药夹心式免疫反应模型中免疫半抗原分子设计提供科学指导依据。

非竞争免疫检测在灵敏度、准确度和检测范围等方面具有较大优势，但小分子农药免疫活性位点单一，难以实现对其的非竞争免疫检测。本项目在前期研究的基础上，以化学结构具有典型研究意义的毒死蜱为研究对象，化学合成粒径为40-100 nm的氧化锆纳米颗粒，用于吸附毒死蜱分子，并借助质谱优化氧化锆对毒死蜱分子的吸附条件；利用化学方法从毒死蜱分子的不同方位引入连接臂，合成了分别不同程度地暴露毒死蜱分子特征结构（免疫活性位点，包括乙氧基硫代磷酸酯结构和芳香环结构）的5种半抗原，制备人工抗原进行免疫；测定抗体的亲和性、特异性，研究半抗原结构对抗体识别取向规律的影响，揭示不同人工抗原下抗体与被氧化锆吸附的毒死蜱分子的识别机理，确立半抗原在充分暴露毒死蜱芳香环结构时更有利于相应的抗体对被氧化锆吸附的毒死蜱分子的识别，并构建以氧化锆为吸附载体的毒死蜱非竞争免疫检测模型。本项目的开展，为毒死蜱非竞争免疫检测提供了基础，同时为有机磷农药的非竞争免疫检测模型的建立提供了科学指导依据。