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

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.

Development of a Biotinylated Nanobody-Based Gold Nanoparticle Immunochromatographic Assay for the Detection of Procymidone in Crops

“…coli BL21. Then the protein in the periplasmic space was extracted by the cold osmotic shock method and then purified using Ni-NTA resin with 200 mmol/L imidazole in 0.01 mol/L PBS for elution . SDS-PAGE analysis of the purified Nbs revealed a protein band slightly higher than 15 kDa, consistent with the expected molecular weight of 18 kDa (Figure b).…”

“…Then the protein in the periplasmic space was extracted by the cold osmotic shock method and then purified using Ni-NTA resin with 200 mmol/L imidazole in 0.01 mol/L PBS for elution. 20 SDS-PAGE analysis of the purified Nbs revealed a protein band slightly higher than 15 kDa, consistent with the expected molecular weight of 18 kDa (Figure 2b). However, the expression levels of Nbs were relatively low, ranging from 0.3 to 1.8 mg/L (Figure 2c), with NbFM3 exhibiting the highest expression level.…”

“…The Nb with the highest affinity was selected. The VHH gene was amplified and ligated to the pINQ-BtH6 vector containing the biotin gene by enzyme digestion and ligation . The recombinant plasmid was transformed into pCY216 natural competence E.…”

“…After the gene library was obtained, a portion of the cells was cultured and infected by M13K07 helper phages to generate a phage library . The genes of PRM selective clones were selected by a simultaneous competition panning strategy, and the screening was performed by ic-ELISA. All candidate clones were sequenced to identify their amino acid sequence.…”

“…The VHH gene was amplified and ligated to the pINQ-BtH6 vector containing the biotin gene by enzyme digestion and ligation. 20 The recombinant plasmid was transformed into pCY216 natural competence E. coli BL21(DE3). Then, the btNb was expressed, purified, and identified via ELISA.…”