Abstract：
A heavy-chain antibody (VHH) library against procymidone (PRM) was constructed via immunizing Bactrian camels. Through careful biopanning, seven nanobodies (Nbs) with different sequences were obtained. The variability in their performance was primarily attributed to the amino acid differences in complementarity-determining region 3 (CDR3), as analyzed by molecular docking. The Nb exhibiting the highest sensitivity, named NbFM5, was biotinylated and conjugated to streptavidin-labeled gold nanoparticles to preserve the epitope\'s activity and prevent a decrease in sensitivity due to traditional random electrostatic adsorption. Subsequently, a simple and sensitive immunochromatographic assay (ICA) was developed for rapid detection of PRM based on biotinylated Nb (btNb). The developed btNb-ICA showed a cut-off value of 200 ng/mL for visual judgment and a half-inhibitory concentration (IC50) of 6.04 ng/mL for quantitative detection. The limit of detection (LOD) was as low as 0.88 ng/mL. The recoveries in actual samples of crops ranged from 82.2 to 117.3%, aligning well with the results obtained from GC-MS/MS (R2 = 0.995). In summary, the developed btNb-ICA demonstrated high specificity and good accuracy for the rapid detection of PRM residues in vegetables. The total analysis time from preparing the sample to obtaining the result was less than 25 min.
摘要：
通过免疫双峰骆驼，构建了针对原嘧啶酮（PRM）的重链抗体（VHH）文库。通过仔细的生物淘选，获得了具有不同序列的七个纳米抗体(Nbs)。其性能的可变性主要归因于互补决定区3（CDR3）的氨基酸差异，通过分子对接分析。Nb表现出最高的灵敏度，命名为NbFM5，被生物素化并与链霉亲和素标记的金纳米颗粒缀合，以保持表位的活性并防止由于传统的随机静电吸附而导致的灵敏度降低。随后，开发了一种基于生物素化Nb（btNb）的快速检测PRM的简单而灵敏的免疫色谱法（ICA）。开发的btNb-ICA显示出用于视觉判断的200ng/mL的截断值和用于定量检测的6.04ng/mL的半抑制浓度(IC50)。检测限(LOD)低至0.88ng/mL。作物实际样品的回收率为82.2％至117.3％，与GC-MS/MS（R2=0.995）获得的结果一致。总之,开发的btNb-ICA对快速检测蔬菜中的PRM残留具有很高的特异性和良好的准确性。从制备样品到获得结果的总分析时间小于25分钟。
