Directed Design of Anti-Idiotypic Nanobody with Antibacterial Activity against Foodborne Staphylococcus aureus and Its Innovative Application in Green Immunoassay

Anti-idiotypic antibodies (Anti-Ids) possess the properties to mimic the structure and biological activity of an antigen, which can be utilized for preventing and monitoring hazards. In this study, Nb4Mutant6-Anti-Id, which mimics the structure and antibacterial activity of vancomycin, was designed based on phage display antibody library screening and mutagenesis technology. The affinity of Nb4Mutant6-Anti-Id for the coated antigens of Van-pAbs F(ab)2 and inactivated S. aureus cells was 6.06 × 10¹⁰ and 2.97 × 10⁸ L/mol, respectively. Nb4Mutant6-Anti-Id maintained approximately 70% of its binding activity for the coated antigens that had undergone heat treatment at 65 °C, and its minimum inhibitory concentration (MIC) against foodborne S. aureus was 105 μg/mL. The limit of detection (LOD) of Nb4Mutant6-Anti-Id protein was 0.72 ng/mL when utilized as a "coated antigen" in a vancomycin-free indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for detecting vancomycin, which is a highly sensitive level (<1.0 ng/mL). This study not only designed Nb4Mutant6-Anti-Id, which exhibits antibacterial activity comparable to that of vancomycin and offering potential for the environmentally friendly management of foodborne pathogens, but also has the potential to replace the vancomycin structure. This replacement enables the establishment of an antigen-free, highly sensitive IC-ELISA with good accuracy and stability for the green monitoring of vancomycin in agricultural environments and food.