Competitive and noncompetitive immunoassays for the detection of benzothiostrobin using magnetic nanoparticles and fluorescein isothiocyanate-labeled peptides

Authors: CHEN, HE - Nanjing Agricultural University; YANG, QIAN - Nanjing Agricultural University; DING, YUAN - Nanjing Agricultural University; VASYLIEVA, NATALIA - University Of California, Davis; Bever, Candace; HUA, XIUDE - Nanjing Agricultural University; WANG, MINGHUA - Nanjing Agricultural University; HAMMOCK, BRUCE - University Of California, Davis

Submitted to: Analytical and Bioanalytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/6/2018
Publication Date: 11/26/2018
Citation: Chen, H., Yang, Q., Ding, Y., Vasylieva, N., Bever, C.R., Hua, X., Wang, M., Hammock, B.D. 2018. Competitive and noncompetitive immunoassays for the detection of benzothiostrobin using magnetic nanoparticles and fluorescein isothiocyanate-labeled peptides. Analytical and Bioanalytical Chemistry. 411:527-535. https://doi.org/10.1007/s00216-018-1478-8.
DOI: https://doi.org/10.1007/s00216-018-1478-8

Interpretive Summary: This study describes the development of an immunoassay for detecting the new fungicide, benzothiostrobin. Phage-displayed peptides were previously isolated that selectively bind the anti-benzothiostrobin monoclonal antibody (mAb) and the benzothiostrobin-mAb immunocomplex. In this study, the peptides were synthesized free of the bacteriophage and conjugated directly to fluorescein isothiocyanate (FITC). The peptide-FITC reagents were used to produce a signal relative to the amount of benzothiostrobin being detected, either in a competitive or noncompetitive assay format. Both formats were used to detect benzothiostrobin in the ng mL-1 (ppb) range and showed good correlation with high-performance liquid chromatography (HPLC) for the detection of benzothiostrobin in authentic samples.

Technical Abstract: Phage-displayed peptides have been proven powerful reagents for competitive and noncompetitive immunoassays. However, they are unconventional reagent, which greatly limits their analytical commercial applications and requires additional reagents for detection. In this work, the peptides that specifically bound with anti-benzothiostrobin monoclonal antibody (mAb) or benzothiostrobin-mAb immunocomplex were synthesized and conjugated with fluorescein isothiocyanate (FITC) as substitutes of the phage-displayed peptides to avoid their shortcomings and extend their applications. Competitive and noncompetitive fluorescence immunoassays (FIAs) for benzothiostrobin were developed by mAb coupling with magnetic nanoparticles as concentration elements and peptides conjugated with FITC as tracers. The competitive FIA showed the half maximal inhibition concentration (IC50) of 16.8 ng mL-1 and detection range (IC10-IC90) of 1.0-759.9 ng mL-1, while the concentration of analyte producing 50% saturation of the signal (SC50) and detection range (SC10-SC90) of noncompetitive FIA were 93.4 and 5.9-788.2 ng mL-1, respectively. The average spiked recoveries were 68.33%-98.50% and 73.33%-96.67% for competitive and noncompetitive FIAs, respectively. The FIAs showed good correlation with high-performance liquid chromatography (HPLC) for the detection of benzothiostrobin in authentic samples.