Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510642, China.
Guangzhou Institute of Food Inspection, Guangzhou 510080, China.
Food Chem. 2024 Oct 15;455:139684. doi: 10.1016/j.foodchem.2024.139684. Epub 2024 May 17.
To break through the bottleneck in preparation of nanobody (Nb) for chemical contaminants induced by the difficulties in the synthesis of immunogen, complexity and unexpectable efficiency of immunization, a novel strategy to generate Nbs based on the designed synthetic Nb libraries with final size up to 10 cfu/mL was adopted and succeeded in selection of anti-coumaphos Nb A4. Furthermore, an affinity-matured mutant Nb 3G was obtained from the secondary library. Finally, an ic-ELISA was established with the limit of detection for coumaphos low to 1.90 ng/mL, 6.4-fold improved than the parent Nb A4, and the detection range from 3.06 to 15.77 ng/mL. Meanwhile, the recovery rate of vegetable samples was from 89.9% to 98.5%. Finally, the accuracy was testified by the standard UPLC-MS/MS method with R up to 0.99. Overall, fully synthetic Nb libraries constructed in this work provided an alternative possibility to generate the specific Nbs for chemical contaminants.
为了突破纳米抗体（Nb）制备中因免疫原合成困难、免疫复杂性和不可预测效率而导致的化学污染物瓶颈，本研究采用了一种新的策略，基于最终大小达到 10 cfu/mL 的设计合成的 Nb 文库来产生 Nb，成功地从抗除草膦的 Nb A4 中筛选出来。此外，从次级文库中获得了亲和力成熟的突变体 Nb 3G。最后，建立了一种 ic-ELISA，其检测限对除草膦低至 1.90ng/mL，比母体 Nb A4 提高了 6.4 倍，检测范围为 3.06 至 15.77ng/mL。同时，蔬菜样品的回收率为 89.9%至 98.5%。最后，通过标准的 UPLC-MS/MS 方法进行了准确性验证，R 高达 0.99。总的来说，本工作构建的完全合成 Nb 文库为生成针对化学污染物的特异性 Nb 提供了一种替代可能性。
