玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇双特异性抗体的制备及检测方法构建

Preparation of Bispecific Antibodies Against Zearalenone and Deoxynivalenol and Construction of Detection Methods

摘要:

玉米赤霉烯酮（zearalenone，ZEN）和脱氧雪腐镰刀菌烯醇（deoxynivalenol，DON）是粮食中常见的真菌毒素，长期的饮食暴露会对生物体造成严重危害。目前针对ZEN和DON同步免疫检测分别依赖各自对应的单克隆抗体，而传统单克隆抗体制备周期长、成本高。作者利用重组抗体表达技术在短时间内获得了可同时识别ZEN和DON的双特异性抗体（Bis-scFv），并构建了基于Bis-scFv的间接竞争酶联免疫检测方法（indirect competitive enzyme-linked immunosorbent assay，IC-ELISA），获得ZEN和DON标准曲线的半数抑制质量浓度（IC₅₀）分别为20.64 ng/mL和132.29 ng/mL；Bis-scFv具有良好的特异性，与其他真菌毒素均无显著的交叉反应。同时，将IC-ELISA方法对玉米中的ZEN和DON进行加标回收实验，其回收率为86.02%～108.14%。本研究证明了所开发的Bis-scFv未来可应用于粮食样本中ZEN与DON的同步快速检测方法的开发。

Abstract:

Zearalenone (ZEN) and deoxynivalenol (DON) are common mycotoxins found in grain, and long-term dietary exposure can cause serious harm to organisms. Currently, simultaneous immunoassays for ZEN and DON mostly rely on their respective monoclonal antibodies. However, the preparation of traditional monoclonal antibodies requires long production cycles and high costs. In this study, bispecific antibodies(Bis-scFv) that could simultaneously recognize both ZEN and DON were obtained in a short time using recombinant antibody expression technology. An indirect competitive enzyme-linked immunosorbent assay(IC-ELISA) based on Bis-scFv was established. The half-maximal inhibitory concentrations(IC₅₀) for ZEN and DON standard curves was determined to be 20.64 ng/mL and 132.29 ng/mL, respectively. Bis-scFv exhibited excellent specificity with no significant cross-reactivity with other mycotoxins. Moreover, IC-ELISA was used to conduct spike recovery experiments for ZEN and DON in corn, yielding recovery rates ranging from 86.02% to 108.14%. The study demonstrates that the developed Bis-scFv has the potential for future application in the development of synchronous and rapid detection of ZEN and DON in grain samples.