Development of a monoclonal-based ic-ELISA for the determination of kitasamycin in animal tissues and simulation studying its molecular recognition mechanism

Abstract

To monitor the residue of kitasamycin (KIT), a monoclonal antibody against KIT was prepared, and a 50% inhibition concentration (IC50) of 5.7 ± 1.4 μg/L was achieved with the most sensitive antibody, KA/2A9, by optimizing ELISA conditions. The LODs for KIT in different animal tissues ranged from 22.47 μg/kg to 29.32 μg/kg, and the recoveries of the fortified tissues were 70% ~ 120% with coefficients of variation below 20%. Then, KIT-specific scFv KA/2A9/3 was prepared for the first time. Homologous modeling and molecular docking results indicated that the key amino acids of KA/2A9/3 scFv are TYR-92 (CDRL3), SER-93 (CDRL3), ASP-155 (CDRH1) and GLY-226 (CDRH3), and the hydrogen bond is the main force. And then, virtual mutation provides a method to evolve KA/2A9/3 scFv antibodies. These results contribute to comprehending the antigen–antibody binding mechanism and provide effective information for in vitro affinity maturation of anti-KIT scFv.