赭曲霉毒素A（OTA）是一种常见的食品污染物，对人和动物具有多种生物毒性作用。酶联免疫吸附分析（ELISA）和胶体金免疫层析（CGI）是OTA例行筛查中常用的两种方法：ELISA的检测灵敏度高，但操作相对繁琐耗时；CGI具有操作简单、检测快速、裸视判读结果等优点，但检测灵敏度相对偏低。而基于酶触发点击化学的等离子体ELISA（ECC-pELISA）兼具ELISA和CGI的优点，制备高亲和力的检测抗体是实现进一步提高其检测灵敏度的关键。本项目拟在已获得的抗OTA纳米抗体（Nb）的基础上，采用同源建模和分子对接技术探析抗OTA Nb的分子识别机理，并通过定向改造获得具有更高亲和力的Nb，进而制备纳米抗体-碱性磷酸酶融合蛋白并探讨其运用于构建ECC-pELISA生物传感体系的可行性。本研究能为其他基因工程抗体的定向分子改造提供模式依据，并为其他小分子化合物的快速、可视化、高灵敏检测提供思路。

Ochratoxin A (OTA) is a common food contaminant, and it can cause multiple biological toxities to human beings and animals. Enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography (CGI) are commonly used for the routine screening of OTA. ELISA has the advantage of high detection sensitivity, but its operation is relatively complicated and time-consuming. CGI has the advantages of easy operation, fast detection and result analysis by naked eyes, while its detection sensitivity is relatively low. Enzyme-triggered click chemistry-plasmonic ELISA (ECC-pELISA) has the advantages of both methods, and the preparation of detection antibody with higher affinity is a key point to further improve its detection sensitivity. In this proposal, the molecular recognition mechanisms of anti-OTA nanobody (Nb) prepared in our previous work will be studied with homology modeling and molecular docking. Nb with improved affinity will be developed by directed modification and then applied to construct nanobody-alkaline phosphatase (Nb-ALP) fusion protein. The feasibility of Nb-ALP fusion protein’s application to establish an ECC-pELISA biosensing system will be further investigated. This study provides a model basis of directed modification for engineered antibodies, and also a new approach for fast, visual and highly sensitive detection of small molecular compounds.