Title: Development of a Highly Sensitive Direct Competitive Fluorescence Enzyme Immunoassay Based on a Nanobody-Alkaline Phosphatase Fusion Protein for Detection of 3-Phenoxybenzoic Acid in Urine.

Authors: Huo, Jingqian; Li, Zhenfeng; Wan, Debin; Li, Dongyang; Qi, Meng; Barnych, Bogdan; Vasylieva, Natalia; Zhang, Jinlin; Hammock, Bruce D

Published In J Agric Food Chem, (2018 Oct 31)

Abstract: 3-Phenoxybenzoic acid (3-PBA) is a human urinary metabolite of many pyrethroid insecticides and can be used as a biomarker to monitor human exposure to these pesticides. A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for detecting 3-PBA on the basis of a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The anti-3-PBA Nb-AP fusion protein was expressed and purified. The 50% inhibitory concentration (IC50) and linear range of dc-FEIA were 0.082 and 0.015-0.447 ng/mL, respectively, with a detection limit of 0.011 ng/mL. The IC50 of dc-FEIA was improved by nearly ten times compared with those of one-step and three-step direct competitive enzyme-linked immunosorbent assay (dc-ELISA). Spiked urine samples were detected by both dc-FEIA and liquid chromatography-mass spectrometry (LC-MS), and the results showed good consistency between the two analysis methods, indicating the reliability of dc-FEIA based on the Nb-AP fusion protein for detecting 3-PBA in urine.

PubMed ID: 30293433

MeSH Terms: Alkaline Phosphatase/chemistry*; Benzoates/urine*; Biomarkers/urine; Enzyme-Linked Immunosorbent Assay; Humans; Immunoenzyme Techniques*; Pesticide Residues/urine*; Pyrethrins/urine; Recombinant Fusion Proteins/chemistry; Single-Domain Antibodies/chemistry*