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

Authors :
Dong J
Li Z
Wang Y
Jin M
Shen Y
Xu Z
Abd El-Aty AM
Gee SJ
Hammock BD
Sun Y
Wang H
Source :
Food chemistry [Food Chem] 2021 Feb 01; Vol. 337, pp. 127780. Date of Electronic Publication: 2020 Aug 05.
Publication Year :
2021

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 <subscript>H</subscript> and V <subscript>L</subscript> 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 <subscript>H</subscript> and V <subscript>L</subscript> 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 (IC <subscript>50</subscript> ) 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.<br />Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.<br /> (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Details

Language :
English
ISSN :
1873-7072
Volume :
337
Database :
MEDLINE
Journal :
Food chemistry
Publication Type :
Academic Journal
Accession number :
32799164
Full Text :
https://doi.org/10.1016/j.foodchem.2020.127780