Abstract: To obtain high-affinity anti-ochratoxin A (OTA) nanobodies, the pET22b-OTA recombinant expression system was utilized to prepare nanobodies through isopropyl-β-D-thiogalactoside (IPTG) induction and periplasmic purification, and the obtained antibody was characterized. Molecular docking was used to analyze the key amino acid sites for the binding of the antibody to the toxin. The results showed that molecular mass of the nanobody was approximately 14 kDa. Its half maximal inhibitory concentration (IC50) value was 1.040 ng/mL, and its cross-reaction rates with the mycotoxins aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) were both below 30%. The antibody remained stable at 25 to 95 ℃, at pH 7.4, at an ion concentration of 25 mmol/L, in 10% to 80% methanol solutions, and in acetone solutions. Molecular docking results confirmed that amino acid residues such as LYS45 and GLU48 mediated high-affinity binding through hydrogen bonds, π-π stacking, and electrostatic interactions. The high-affinity anti-OTA nanobody provides a high-performance molecular tool for food safety detection, demonstrating significant application value for ensuring food safety.

Key words: ochratoxin A; nanobody; antibody characteristics; molecular docking