Development of a Nanobody-Alkaline Phosphatase Fusion Protein and Its Application in a Highly Sensitive Direct Competitive Fluorescence Enzyme Immunoassay for Detection of 3-Phenoxybenzoic Acid in Urine

摘要

3-Phenoxybenzoic acid (3-PBA), >1() 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 3-PBA based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The anti-3-PBA Nb-AP fusion protein was expressed and purificated. The 50% inhibitory concentration (IC50) and the detection limit of the dc-FEIA were 0.082 and 0.011 ng/mL, respectively, with a linear range of 0.015–0.447 ng/mL. The IC50 of the one-step dc-FEIA was improved by nearly ten times compared with that of the one-step and three-step dc-ELISA. This assay was also compared with LC-MS for detecting the spiked urine samples, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring 3-PBA in urine.Structure of 3-phenoxybenzoic acid, >1, and pyrethroids containing a phenoxybenzyl group, >2–6.