Biotin-labelled peptidomimetic for competitive time-resolved fluoroimmunoassay of benzothiostrobin.

In recent years, more and more functional peptide ligands have been identified from phage display libraries and served the immunoassay of small molecules. After the identification, the phage particle instead limits further application of peptide ligands, so it is of great significance to explore the peptide ligand as an independent detection reagent. In this work, the identified peptidomimetic of benzothiostrobin was synthesized and labelled with biotin, which was combined with Eu3+-labelled streptavidin to develop the peptide-based time-resolved fluoroimmunoassay (P-TRFIA). Under the optimal conditions, the half-maximum inhibitory concentration (IC50) of proposed P-TRFIA is 3.63 ng mL-1, which is similar to the TRFIA using phage-borne peptidomimetic and Eu3+-labelled anti-phage antibody (IC50: 4.55 ng mL-1), also more sensitive than previously reported immunoassays for benzothiostrobin. In addition, the proposed P-TRFIA shows excellent specificity and accuracy for analysis of spiked samples, and its detection results shows good consistency with high-performance liquid chromatography for the detection of environment and agro-products samples with unknown benzothiostrobin concentrations.

Competitive and noncompetitive fluorescence polarization immunoassays for the detection of benzothiostrobin using FITC-labeled dendrimer-like peptides.

Peptides obtained from phage display libraries are valuable reagents for small-molecule immunoassays. However, their application in fluorescence polarization immunoassays (FPIAs) is limited by phage particles. Here, monomer, dendrimer-like dimer, tetramer peptidomimetic and anti-immunocomplex tracers were designed and synthesized using lysine as special scaffolds and spacers to develop competitive and noncompetitive FPIAs for benzothiostrobin. The affinity between tracers and monoclonal antibodies or immunocomplexes increased with the tracer valence. A higher signal-to-noise ratio and sensitivity could be generated in the FPIAs based on tetramer tracers. The sensitivities of competitive (50% inhibitory concentration) and noncompetitive (50% saturation concentration) FPIAs were 19.71 ± 4.65 and 40.43 ± 2.73 ng mL-1, respectively. The spiked recoveries were 78.3%-105.2% with relative standard deviations (RSDs) of 0.7%-15.4% for the competitive FPIA, while 78.7%-115.3% with RSDs of 0.7%-12.5% for the noncompetitive FPIA. The amounts of benzothiostrobin in rice detected by the FPIAs were consistent with those detected by high performance liquid chromatography.