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

文献类型： 外文期刊

第一作者： Dong, Jiexian

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

作者机构：

关键词： Database-assisted sequence analysis; ScFv-AP based immunoassay; Malachite green; Residue determination; Tilapia fish

期刊名称：FOOD CHEMISTRY （ 影响因子：7.514； 五年影响因子：7.516 ）

ISSN： 0308-8146

年卷期： 2021 年 337 卷

页码：

收录情况： SCI

摘要： 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.

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