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

To determine malachite green (MG) and its major metabolite, leucomalachite (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 VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against LMG, then thoroughly by database-assisted sequence analysis. Finally, the productive assembled to an intact scFv engineered produce scFv-AP The protein further identified as bifunctional reagent for immunoassay, sensitive one-step CLEIA LMG with half-maximal inhibitory concentration (IC50) limit of detection (LOD) 1.3 0.04 ng/mL, respectively. validation results this novel competitive line those obtained classical HPLC method determination total MG spiked field incurred samples.