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, ABDELATY; Gee, Shirley; Hammock, Bruce; Sun, Yuanming; Wang, Hong

doi:10.1016/j.foodchem.2020.127780

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 J., Li Z., Wang Y., Jin M., Shen Y., Xu Z., ...Daha Fazla

Food Chemistry, cilt.337, 2021 (SCI-Expanded, Scopus)

Yayın Türü: Makale / Tam Makale
Cilt numarası: 337
Basım Tarihi: 2021
Doi Numarası: 10.1016/j.foodchem.2020.127780
Dergi Adı: Food Chemistry
Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, PASCAL, Aerospace Database, Aquatic Science & Fisheries Abstracts (ASFA), BIOSIS, CAB Abstracts, Chemical Abstracts Core, Chimica, Communication Abstracts, Compendex, EMBASE, Food Science & Technology Abstracts, MEDLINE, Metadex, Veterinary Science Database, Civil Engineering Abstracts
Anahtar Kelimeler: 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
Atatürk Üniversitesi Adresli: Evet

Özet

© 2020 Elsevier LtdTo 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, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against LMG, and then thoroughly by database-assisted sequence analysis. Finally, the productive VH and VL 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.