Single-chain variable fragment antibody-based ic-ELISA for rapid detection of macrolides in porcine muscle and computational simulation of its interaction mechanism

Abstract

To generate a single-chain fragment variable (scFv) for the detection of macrolide antibiotics (MAs) residues, the biopanning, expression and comparative analysis of four scFvs against MAs generated from five phage display libraries were performed. Three-dimensional (3D) conformation comparative analysis revealed that changes in the active pocket, caused by mutations in the FR framework region, are the main factor affecting the recognition ability of scFv. Homology modeling and molecular docking indicated that the binding sites between the optimal MA-specific scFv candidate DA/3C4/23 and MA are PRO-221, ASP-226, SER-153, PHE-154, ASP-226, and ARG-45. After optimization of soluble expression conditions, stable soluble DA/3C4/23 scFv antibody was obtained, and a 50% inhibition concentration (IC 50 ) of 10.9 μg/L for tylosin (TYL) was achieved. The recoveries of the muscle tissues were 96.73%–107.24% with coefficients of variation below 20%. It indicated that the established method can be effectively applied for food safety monitoring. • Multiple new MAs-specific scFvs were screened from five phage display libraries. • The binding mode between scFv and MAs was first reported. • In-depth comparison of 3D structures between scFvs with different affinities. • The scFv-based ic-ELISA for monitor of MAs in animal tissues was first established.