Antibody Screening and Binding Prediction Analysis Targeting Stx2

Shiga toxin (Stx), produced by enterohemorrhagic <i>Escherichia coli</i> (EHEC), is a highly potent exotoxin responsible for severe complications such as hemolytic uremic syndrome (HUS). Among its isoforms, Stx2 exhibits stronger cytotoxicity and poses greater clinical risk, yet no effective therapy currently exists. In this study, two human monoclonal antibodies, YG12-1 and YG12-2, were identified from a phage-display library and systematically characterized using an integrated modeling–validation workflow. Structural modeling with <i>ImmuneBuilder</i> and <i>Rosetta</i> revealed that YG12-2 possessed a longer CDRH3 topology, more short-range hydrogen bonds, and stronger electrostatic complementarity, corresponding to lower binding energy and higher apparent affinity in ELISA.Despite its superior structural stability, YG12-2 exhibited weaker in vivo protection than YG12-1 in a murine peritoneal infection model, where YG12-1 conferred a dose-dependent survival advantage under lethal <i>E. coli</i> O157:H7 challenge. This paradox highlights a non-linear relationship between structural affinity and biological efficacy, emphasizing the importance of functional epitope accessibility and pharmacokinetic behavior in determining neutralization outcomes.Overall, this study demonstrates a computation-guided framework that bridges structural prediction with experimental validation, providing mechanistic insights into Stx2 recognition and suggesting potential structural and functional factors underlying antibody efficacy against Shiga toxin.