Article 2021 · 54 citations

Generation of functional single-chain fragment variable from hybridoma and development of chemiluminescence enzyme immunoassay for determination of total malachite green in tilapia fish

Dong, Jiexian, Li, Zhenfeng, Wang, Yu, Jin, Maojun, Shen, Yudong, Xu, Zhenlin, Abd El-Aty, A. M., Gee, Shirley J., Hammock, Bruce D., Sun, Yuanming, Wang, Hong

Abstract

To determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) residual levels in tilapia fish, chemiluminescent enzyme immunoassay (CLEIA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. At first, V-H and V-L gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against LMG, and then thoroughly by database-assisted sequence analysis. Finally, the productive V-H and V-L were assembled to an intact scFv sequence and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step CLEIA against LMG was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 1.3 and 0.04 ng/mL, respectively. The validation results of this novel competitive CLEIA was in line with those obtained by classical HPLC method for determination of total MG in spiked and field incurred samples.

Keywords

Database-assisted sequence analysis ScFv-AP based immunoassay Malachite green Residue determination Tilapia fish LEUCOMALACHITE GREEN LIQUID-CHROMATOGRAPHY CRYSTAL VIOLET CELL-LINES CLONING IDENTIFICATION TISSUE ELISA

WoS Categories

Chemistry, Applied Food Science & Technology Nutrition & Dietetics

Citation

Journal: FOOD CHEMISTRY
Year: 2021
Volume: 337
ISSN: 0308-8146
eISSN: 1873-7072
DOI: 10.1016/j.foodchem.2020.127780
WoS UID: WOS:000576952500015

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