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 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.

Automated summary

A chemiluminescent enzyme immunoassay (CLEIA) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed to determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) residual levels in tilapia fish. The validation results of this novel competitive CLEIA were consistent with those obtained by classical HPLC method for determination of total MG in spiked and field incurred samples.