Response surface methodology for optimization of scFv-BAD anti-E2 CHIKV expression in Escherichia coli Origami B (DE3) for the detection of Chikungunya virus.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for recurring outbreaks, highlighting the need for rapid and accurate diagnostic tools. The E2 glycoprotein of CHIKV is a promising target for antibody-based detection. This study aims to determine the optimal expression conditions of recombinant single-chain variable fragment (scFv) fused with biotin acceptor domain (BAD) specific to the CHIKV E2 glycoprotein in Escherichia coli Origami B (DE3). The optimization was performed using the Box-Behnken design of response surface methodology (RSM), with the IPTG inducer concentration, induction time, and induction temperature as the independent variables. The optimal conditions were identified as 0.2 mM IPTG, 2 hours induction at 37 °C, resulting in a total protein concentration of 0.658 mg/mL. The soluble fraction of scFv-BAD was successfully purified using Ni²⁺-NTA affinity chromatography, with a purity of 91.11%. ELISA confirmed that the recombinant scFv-BAD was biotinylated and retained its ability to bind the CHIKV E2 antigen. The optimized scFv-BAD construct demonstrates potential for use in various immunoassay platforms, including rapid diagnostic tests for CHIKV detection.