Affinity maturation of anti-ochratoxin a nanobody via synthetic library strategy for enhanced small molecule detection

Single-domain properties and ease of gene manipulation of the nanobody (Nb) make it suitable for affinity maturation in vitro. In this study, the anti-ochratoxin A (OTA) Nb3G was selected as a model antibody to explore feasible approach of affinity enhancement. Artificial intelligence (AI)-augmented molecular modeling tools were used to analyze the interaction between OTA and Nb3G, and nine key residues (Thr28, Tyr32, Asp54, Thr57, Trp99, Phe103, Glu106, Trp110, Tyr112) were selected to construct a site-directed mutagenesis library. After biological screening and identification, the affinity-enhanced mutants 1H and 11B were obtained, exhibiting half maximal inhibitory concentration (IC₅₀) values of 0.31 ng/mL and 0.25 ng/mL, corresponding to 1.26-fold and 1.56-fold sensitivity improvements compared with the parent Nb (IC₅₀ 0.39 ng/mL). Enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) further confirmed 2-fold and 4-fold increases in titer, and 6.27-fold and 11.49-fold improvements in affinity, respectively. The mutants also demonstrated superior stability under diverse conditions and improved competitive inhibition performance with lower variability. Structural analysis indicated that deeper antigen-binding cavities may facilitate more efficient OTA encapsulation, enhancing detection sensitivity. Recoveries in spiked oat and coffee samples ranged from 94.20 % to 113.85 %. Overall, the synthetic nanobody library strategy developed in this work provides an effective method for the preparation of high-affinity nanobodies against small molecule antigens.