1. Production and Characterization of Biotinylated Anti-fenitrothion Nanobodies and Development of Sensitive Fluoroimmunoassay
- Author
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Zi-Jian Chen, Yi-Feng Zhang, Jia-Lin Chen, Ze-Shan Lin, Min-Fu Wu, Yu-Dong Shen, Lin Luo, Hong Wang, Xiao-Wei Wen, Bruce Hammock, Hong-Tao Lei, and Zhen-Lin Xu
- Subjects
Agricultural and Veterinary Sciences ,Prevention ,Fluoroimmunoassay ,fenitrothion ,Enzyme-Linked Immunosorbent Assay ,Fenitrothion ,General Chemistry ,Single-Domain Antibodies ,Article ,nanobody ,Engineering ,Tandem Mass Spectrometry ,carbon dots ,Chemical Sciences ,biotinylation ,Streptavidin ,General Agricultural and Biological Sciences ,Biotechnology ,Food Science - Abstract
A simple and sensitive fluoroimmunoassay (FIA) based on a heavy-chain antibody (VHH) for rapid detection of fenitrothion was developed. A VHH library was constructed from an immunized alpaca, and one clone recognizing fenitrothion (namely, VHHjd8) was achieved after careful biopanning. It was biotinylated by fusing with the Avi tag and biotin ligase to obtain a fusion protein (VHHjd8-BT), showing both binding capacity to fenitrothion and the streptavidin poly-horseradish peroxidase conjugate (SA-polyHRP). Based on a competitive assay format, the absorbance spectrum of oxidized 3,3',5,5'-tetramethylbenzidine generated by SA-polyHRP overlapped the emission spectrum of carbon dots, which resulted in quenching of signals due to the inner-filter effect. The developed FIA showed an IC50 value of 1.4 ng/mL and a limit of detection of 0.03 ng/mL, which exhibited 15-fold improvement compared with conventional enzyme-linked immunosorbent assay. The recovery test of FIA was validated by standard GC-MS/MS, and the results showed good consistency, indicating that the assay is an ideal tool for rapid screening of fenitrothion in bulk food samples.
- Published
- 2022