Development of a sensitive direct competitive chemiluminescent enzyme immunoassay for gentamicin based on the construction of a specific single-chain variable fragment-alkaline phosphatase fusion protein

Weijie Deng; Dan Wang; Peng Dai; Yanping Hong; Jianhua Xiong; Luying Duan; Ruimin Lu; Jianchun Wan; Huaying Du; Bruce D Hammock; Wuying Yang

doi:10.1016/j.microc.2023.109706

Development of a sensitive direct competitive chemiluminescent enzyme immunoassay for gentamicin based on the construction of a specific single-chain variable fragment-alkaline phosphatase fusion protein

Microchem J. 2024 Feb:197:109706. doi: 10.1016/j.microc.2023.109706. Epub 2023 Nov 30.

Authors

Weijie Deng¹, Dan Wang¹, Peng Dai¹, Yanping Hong¹, Jianhua Xiong¹, Luying Duan¹, Ruimin Lu¹, Jianchun Wan², Huaying Du¹, Bruce D Hammock³, Wuying Yang^{1

3}

Affiliations

¹ Key Laboratory of Agricultural Products Processing and Quality Control of Nanchang City/College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.
² Technology Center of Nanchang Customs District, Nanchang 330038, China.
³ Department of Entomology and Nematology and UCD Comprehensive Cancer Center, University of California, Davis, CA 95616, United States.

PMID: 38283367
PMCID: PMC10810264 (available on 2025-02-01)
DOI: 10.1016/j.microc.2023.109706

Abstract

A sensitive chemiluminescent enzyme immunoassay (CLEIA) was established for the determination of gentamicin (GEN) residue levels in animal tissue. This assay is based on a fusion protein of single-chain variable fragment (scFv) and alkaline phosphatase (AP). Initially, V_L and V_H derived from anti-gentamicin monoclonal antibody were linked by a short peptide to construct a scFv. Subsequently, the constructed scFv sequence was accessed into the pLIP6/GN vector, and a soluble scFv-AP fusion protein was generated. The scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) for the determination of gentamicin. In the dcCLEIA, the half inhibitory concentration (IC₅₀) and limit of detection (LOD) were 1.073 ng/mL and 0.380 ng/mL, respectively. The average recoveries of gentamicin spiked in animal tissue samples ranged from 78% to 96%. These results showed a strong correlation with ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The above results suggest that the anti-GEN scFv-AP fusion protein is suitable for detecting gentamicin residues in edible animal tissues.

Keywords: Alkaline phosphatase; Anti-gentamicin scFv; Chemiluminescent enzyme immunoassay; Gentamicin; ScFv-AP fusion protein.

Abstract

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