基于双功能抗体的农药多残留免疫检测技术的基础研究

In the recent years, immunoassays have known as rapid and reliable analytical techniques for pesticides detection. However, most of the immunoassays developed were used to detect individual pesticide residue, few for multi-pesticide residues due to their narrow detection range and low sensitivity. In this study, the engineered antibody technology was used as a new strategy to develop immunoassays for multi-pesticide residues. First, two kinds of generic haptens that preserve the common structure of phosphate ester pesticides and thiophosphate ester pesticides, respectively, were synthesized and conjugated with carrier proteins, and then BALB/C mice were immunized with selected immunogens and broad specificity single chain variable fragments (scFv) antibodies specific to two types of organophosphorus pesticides as described above were produced with phage display technology. Second, the scFv antibodies were mutated with in vitro maturation technologies for the improvement of detection range and sensitivity. And then, the bispecific diabodies were constructed with the cross pair of VL domain and VH domain of different of broad specificity scFc antibodies for further improvement of the detection ranges. This study will provide scientific basis for development of broad specific, high sensitive,and rapid immunoassay to multi- pesticide residues.

目前免疫检测作为快速可靠的检测技术在农药单残留检测上应用较多，而在农药多残留检测的运用上受到限制，困扰这一问题的关键是方法的检测谱窄、灵敏度不足。我们研究发现虽然通用结构半抗原和多簇人工抗原的引入，能够在一定程度上提高农药多残留免疫分析的检测谱，但不能从根本上解决以上问题。因此，本研究拟利用基因工程抗体技术，提出解决以上问题的新思路。本研究拟首先合成代表磷酸酯类和硫代磷酸酯类两类有机磷农药的通用结构半抗原；在此基础上利用噬菌体展示技术制备分别针对以上两类有机磷农药的"宽谱特异性"单链抗体；并利用抗体体外成熟技术对单链抗体进行改造，以提高免疫检测的"灵敏度"和"检测谱"；然后，将不同宽普特异性识别的单链抗体的轻、重链可变区基因交叉配对组装于同一载体，制备双功能抗体，使其能同时识别以上两类有机磷农药，以进一步扩大"检测谱"。该研究将为建立检测谱宽、灵敏度高的农药多残留快速检测技术提供科学依据。

有机磷农药广泛用于蔬菜、水果、农作物等的病害虫防治。由于使用量大，有机磷农药的残留已经成为一个严重问题，通过污染自然环境对养殖动物和人类造成较大威胁。此外，这类农药的残留还严重影响了我国茶叶、农作物、蔬菜、水果等物资的出口。因此开展有机磷农药多残留检测技术与检测机理研究具有重要的意义。本研究制备了针对有机磷农药的宽谱特异性单克隆抗体、单链抗体，并利用随机突变技术制备了突变抗体；在此基础上制备了双功能抗体，并进一步探讨了抗体-抗原的作用机制。具体研究结果如下：根据有机磷农药的共有结构，合成了22种半抗原，并与载体蛋白连接形成人工抗原；免疫Balb/C小鼠后，通过细胞融合、克隆化，共获得11株特异性较好的杂交瘤细胞克隆，并对单克隆抗体特性进行了研究；从单克隆杂交瘤细胞和免疫后的小鼠脾脏细胞构建了噬菌体展示抗体库，并从中筛选出了分别针对O，O-二甲氧基类和O，O-二乙氧基类有机磷农药的单链抗体；其中针对O，O-二乙氧基类有机磷农药的单链抗体可以识别32种有机磷农药，与其母本杂交瘤细胞分泌的单克隆抗体的识别谱相当，但其与有机磷农药的交叉反应率比母本单克隆抗体更均匀；以获得的单链抗体基因为模板，利用易错PCR构建了突变抗体库，从中筛选出的O，O-二甲氧基类有机磷农药的单链抗体的亲和力和识别谱有了一定的提高。以获得的单链抗体为基础，通过不同单链抗体的轻、重链可变区基因交叉配对在大肠杆菌细胞内表达，细胞外复性组装的方法获得了双功能抗体，对抗体活性检测显示，获得的双功能抗体保持了原有的单链抗体的活性。本研究的开展，对小分子危害物免疫检测技术的发展与免疫机理的阐明起到了推动作用。