基于半抗原与抗原构象关系的食用油外源标志物通用抗体识别机制研究

The adulteration of waste cooking oil in qualified edible oil has already been the severe problem of harming public health, and seldom effective detection method has been devoted to this questions. Development of rapid immunoassays for simultaneous detection of foreign markers, like capsaicinoids and gingerol, in waste cooking oil can effectively solve this problem, and highly sensitive and broad-specificity antibody is the key reagents. Our previous studies show that the capsaicinoids haptens with different structure have a significant effect on the immunoassay method, but the specific recognition mechanism of antibody is not clear. Thus, this project intends to design and synthesize several kinds of haptens with different spacer-arms, product the corresponding antibodies, and compare the specificity of these antibodies under homologous and heterologous conditions. The conformational change model of hapten-protein conjugate could be established by molecular simulation, and the effect of conformation on antibody characteristics will be explained. To investigate the molecular mechanisms that affect antibody sensitivity, homology modeling and molecular docking will be performed in this study. In conclusion, the research of this project will provide clues into the interactions between broad-specificity antibody and hapten, which will guide future modifications of haptens and production of antibodies against other foreign markers in edible oils.

由餐厨废弃油脂引起的食用油掺伪问题危害重大，目前仍缺乏快速识别与鉴定方法。经研究调查发现，建立餐厨废弃油脂外源标志物辣椒素及姜辣素等物质同步免疫分析技术是解决这一问题的有效途径，而高灵敏且通用型抗体是其关键试剂。申请人前期研究表明，不同结构辣椒素类物质半抗原对免疫分析方法影响显著，但抗体多特异性识别机制不明确。因此，本项目拟补充设计合成不同连接臂的半抗原，研制出相应的抗体，并对比同源与异源条件下通用抗体识别规律；借助分子模拟手段建立人工抗原构象变化模型，分析半抗原/人工抗原空间结构取向对抗体性能的影响；综合采用同源建模、分子对接等方法研究通用性抗体与靶标分子复合物的稳定空间结构形式，确定通用抗体关键识别位点,揭示半抗原/人工抗原构象关系对通用抗体识别特性的影响规律，为食用油中多种小分子外源标志物同步免疫分析提供理论依据和技术支撑。

餐厨废弃油脂问题是食用油质量安全领域面临的重大难题。本项目以餐厨废弃油脂外源标志物辣椒素、姜辣素类物质为目标，补充设计合成了16种连接臂位点、长度、和结构不同的辣椒素、姜辣素类物质半抗原。（1）结合前期ELISA分析结果，选出9种有代表性的半抗原，利用Gaussian软件对其结构最低能量构象和静电势能进行了模拟，选取载体蛋白上的多肽分别与特征半抗原进行模拟偶联，获得通用半抗原-多肽偶联复合物的空间构象，建立“通用半抗原-多肽”模型。（2）利用软件MOEv2018.01对代表性的辣椒素类物质单克隆抗体可变区进行了同源模建，结合半抗原/人工抗原空间结构取向对抗体性能的影响，分析了半抗原/人工抗原构象关系对通用抗体识别特性的影响规律，将所得模型与辣椒素、姜辣素类物质半抗原进行了分子对接，明确了抗体关键氨基酸识别位点。（3）在以上模型及分析结果的指导下，选择通用抗原BSA-hapten TO、BSA-hapten GI和BSA-hapten X’作为免疫抗原研制通用抗体。进一步对比了同源、异源条件下抗原-抗体结合规律，最终确定最佳抗原抗体组合，其对姜辣素、辣椒素类物质的灵敏度分别为：3.73 ng/mL和24.76 ng/mL。（4）对电化学生物传感检测方法进行了系统的研究，通过建立一系列灵敏度高、通用性好且快速简便的电化学生物传感分析方法，确定了食用植物油掺伪标志物辣椒素类物质电化学免疫分析的各项实验条件。（5）采用三电极体系，构建了餐厨废弃油脂标志物的检测模型：基于TH/PB-AuNP双信号扩增的灵敏电化学免疫传感器，用于餐厨废弃油脂的鉴别检测，检出限为0.01 ng/mL，稳定性高；构建的基于Fe3O4NPs@CeO2修饰的竞争型电化学检测平台，利用Fe3O4NPs@CeO2的高电导率和氧化还原性与适当的生物识别元件相结合，进一步提高了免疫传感器的灵敏度、特异性和稳定性。在最佳测试条件下，该免疫传感器的检出限为0.33 pg/mL。本研究为食用油中多种小分子外源标志物同步免疫分析提供理论依据和技术支撑。