基于噬菌体展示纳米抗体的绿色免疫PCR检测脱氧雪腐镰刀菌烯醇

目的：在获得脱氧雪腐镰刀菌烯醇（deoxynivalenol，DON）抗独特型纳米抗体的前期基础上，将纳米抗体作为酶标抗原的替代物，应用于荧光定量免疫聚合酶链式反应（polymerase chain reaction，PCR）体系，实现DON的高灵敏、绿色免疫分析。方法：将特异性结合DON抗体的噬菌体展示纳米抗体（P-28）作为竞争抗原，以编码P-28纳米抗体的DNA为靶标，设计特异性PCR扩增引物，优化荧光定量免疫PCR退火温度、抗DON抗体浓度、P-28投入量等参数，建立基于间接竞争模式的荧光定量免疫PCR检测DON的方法。结果：本研究建立的荧光定量免疫PCR方法线性检测范围为0.1～1 000 ng/mL，IC50值为（3.96±2.21）ng/mL，最低检出限为0.048 ng/mL，并与其他真菌毒素无交叉反应。结论：该方法直接使用噬菌体展示纳米抗体作为竞争抗原的替代物，应用于免疫PCR体系，避免了使用传统的化学合成酶标抗原所带来的环境污染、操作毒性等缺陷，并具有良好的特异性及灵敏度。

Phage Displayed Nanobody Mediated Green Immuno-PCR for Detection of Deoxynivalenol

Objective: In order to develop a highly sensitive and green immunoassay for deoxynivalenol (DON) anti-idiotypic nanobody, obtained in our previous study, was used as an alternative to enzyme-labeled antigen in a fluorescence real-time immuno-PCR system. Methods: The phage displayed nanobody (P-28), which specifically binds with anti-DON antibody, was used as a competitive antigen and the DNA encoding P-28 Nanobody was used as a target for primer design. Important experimental parameters including annealing temperature, anti-DON antibody concentration and P-28 amount were optimized. Finally, we proposed a fluorescence real-time immuno-PCR method based on indirect competition for the detection of DON. Results: The linear range of the immuno-fluorescence PCR method was 0.1–1 000 ng/mL with an IC50 of (3.96 ± 2.21) ng/mL, and the limit of detection (LOD) was 0.048 ng/mL. This method showed no crossreaction with other mycotoxins. Conclusion: This method avoids the defects of environmental pollution and operational toxicity caused by using traditional synthetic enzyme-labelled antigen and has good specificity and sensitivity.