Production and Characterization of Biotinylated Anti-fenitrothion Nanobodies and Development of Sensitive Fluoroimmunoassay

Overview

Journal J Agric Food Chem

Specialties Chemistry
Nutritional Sciences

Date 2022 Mar 25

PMID 35333506

Authors

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

Zhen-Lin Xu

Affiliations

Soon will be listed here.

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 IC 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.

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