基于人工免疫原构象模拟的半抗原设计与高亲和力抗氟乐灵抗体的诱导

Antigen-antibody based immunoassay plays an important role in food contaminants detection of chemical compounds. And antibodies with high quality are crucial for immunoassay establishment. Once hapten coupled with protein, its conformational changes influence the immunogenicity of artificial antigen directly, and the properties of induced antibody would be influenced greatly. Nowadays, most hapten designs are restricted to structures of hapten, but the structure and conformational changes of hapten coupled on protein are rarely mentioned. Therefore, in this study, herbicide trifluralin was chose as an example molecule for high affinity antibody preparation. Based on the primary designed structure of hapten, three dimensional structures of trifluralin-protein conjugates would be built. And molecular dynamics simulation methods would be performed on hapten’s preferential conformation and hapten-protein interaction. The inner link between structure/conformation of immunogen and affinity promotion of induced antibody would also be explored. Then hapten would be redesigned. It is expect to obtain anti-trifluralin antibodies with high affinity, and build super-sensitivity immunoassay for detecting trifluralin residues in sea food. Hapten design method based on conformational simulation of immunogen is of scientific significance for accelerating development of hapten immunoassay with high sensitivity and proper specificity.

基于抗原-抗体反应的免疫分析技术在食品小分子化学污染物残留检测领域占有重要地位，其中高质量抗体的获得是建立免疫分析方法的关键。免疫原上半抗原的结构/构象变化直接影响了目标半抗原的免疫原性，对所诱导的抗体性能有重要影响。目前半抗原设计多基于对半抗原结构本身的设计，而鲜有考虑半抗原与载体蛋白偶联后所发生的构象变化。基于此，本研究以氟乐灵为模型，在半抗原初步设计的基础上，应用分子模拟的方法构建氟乐灵-蛋白偶联物的三维空间结构，并采用分子动力学方法对人工免疫原上氟乐灵的优势构象与相互作用情况进行模拟分析，探讨人工免疫原构象变化与抗体亲和力之间的关系，进而指导半抗原的定向设计，以期获得高亲和力抗氟乐灵抗体，并建立高灵敏度的水产品中氟乐灵残留检测的免疫分析方法。基于人工免疫原构象模拟的半抗原设计方法的实现，对于加快小分子化学污染物高灵敏度和高（或广谱）特异性的免疫分析技术的发展具有重要的科学意义。

氟乐灵农药在农产品中的残留问题已引起人们的广泛关注，其高亲和力抗体制备困难是限制免疫分析作为高通量快速筛查方法应用于氟乐灵检测的主要原因。本研究针对氟乐灵的结构特点，以半经验方法结合电势和构象模拟以及构象叠合等分子模拟手段设计合成了6种半抗原。Balb/c小鼠免疫筛选表明半抗原2C和1C所诱导的抗体具有较高亲和力（IC50分别为2000和8 ng/mL）。制备相应兔多克隆抗体，并建立了以2C诱导的多克隆抗体的免疫分析方法：IC50为12.21 ng/mL，检测限（LOD，IC10）为0.29 ng/mL，线性范围为2.56~58.23 ng/mL。在鳗鱼、对虾、大豆和大豆油4种食品样品的添加回收试验中，icELISA测得氟乐灵的平均回收率为89.8%~106.4%，而GC法测得的平均回收率为89.0%~108.2%。基于1C和2C抗体的交叉反应数据分别建立了CoMSIA和CoMSIA模型，结果显示静电场、氢键供体场以及立体场对抗体亲和力的影响较大。应用细胞融合方法获得5株杂交瘤细胞株，其中细胞株McAb-L001产生的抗体灵敏度最好，IC50为2.05 ng/mL，检测限（LOD，IC10）为0.31 ng/mL，线性范围为 0.55~7.58 ng/mL，与14种氟乐灵相似物的交叉反应性可得其特异性高。氟乐灵高灵敏度免疫分析方法的建立填补了我国氟乐灵农药残留免疫检测领域的空白，为农产品中氟乐灵的检测提供了灵敏、准确、高通量的检测方法。应用构象模拟的半抗原设计策略有助于高亲和力抗氟乐灵抗体的诱导。