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.

Journal: Microchemical journal : devoted to the application of microtechniques in all branches of science
Published Date: Nov 30, 2023

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 and V 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 (IC) 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.

Authors

  • Weijie Deng
    Key Laboratory of Agricultural Products Processing and Quality Control of Nanchang City/College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.
  • Dan Wang
    Guangdong Pharmaceutical University Guangzhou Guangdong China.
  • Peng Dai
    Key Laboratory of Agricultural Products Processing and Quality Control of Nanchang City/College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.
  • Yanping Hong
    Key Laboratory of Agricultural Products Processing and Quality Control of Nanchang City/College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.
  • Jianhua Xiong
    Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin, China.
  • Luying Duan
    Key Laboratory of Agricultural Products Processing and Quality Control of Nanchang City/College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.
  • Ruimin Lu
    Key Laboratory of Agricultural Products Processing and Quality Control of Nanchang City/College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.
  • Jianchun Wan
    Technology Center of Nanchang Customs District, Nanchang 330038, China.
  • Huaying Du
    Key Laboratory of Agricultural Products Processing and Quality Control of Nanchang City/College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.
  • Bruce D Hammock
    Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California, Davis, CA 95616, USA.
  • Wuying Yang
    Key Laboratory of Agricultural Products Processing and Quality Control of Nanchang City/College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.

Keywords

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