The characteristics of single domain and ease of gene manipulation of the single domain antibody (sdAb) make it suitable for affinity maturation in vitro. Since the affinity of antibodies can influence the immunoassays' sensitivity, a nanobody (Nb), the anti-ochratoxin A sdAb (AOA-sdAb), was herein selected as the model antibody to explore feasible approach for improving its affinity. Homology modeling and molecular docking were used to analyze the interaction between OTA and the AOA-sdAb. After alanine scanning verification, Gly53, Met79, Ser102, and Leu149 were determined as the key amino acids of the AOA-sdAb. Two site-directed saturated mutation libraries were constructed by two-site mutation against those four key amino acids. After biopanning and identification, a mutant Nb-G53Q&S102D was obtained with a half maximal inhibition concentration (IC50) of 0.29 ng/mL and a KD value of 52 nM, which is 1.4-fold and 1.36-fold lower than that of the original sdAb, respectively. The computer simulation analysis indicated that the hydrogen bond, hydrophobic interaction, and side chain steric hindrance of amino acid residues are critical for the binding affinity of the AOA-sdAb. Overall, the techniques shown in this study are effective ways at ‘identifying residues involved in antigen binding’ that can be altered by site-directed mutation.
