Tolerance patterns of ochratoxin A nanobody in organic solvents

The extraction of mycotoxins from complex food matrices for immunoassay often requires organic solvents, which can compromise antibody integrity and detection sensitivity. This study aimed to investigate the tolerance patterns of an ochratoxin a (OTA)-specific nanobody in organic solvents, elucidating the relationship between its structural stability and functional activity. Direct competitive enzyme-linked immunosorbent assay, fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy, Fourier transform infrared spectroscopy, and molecular docking were employed to systematically analyze the tolerance thresholds, antibody activity retention rates, and structural changes of the OTA nanobody in eight organic solvents.The results showed that the OTA nanobody exhibited higher tolerance in methanol, ethylene glycol, glycerol, acetone and dimethyl sulfoxide (thresholds of 40%-60%), while lower tolerance was observed in acetonitrile and dimethylformamide (thresholds of 20% and 10%, respectively). Results of antibody activity retention rate and spectral analysis showed that methanol induced minimal structural and functional damage to OTA nanobodies; acetone severely impaired the nanobodies' structure and activity with significant destruction of their core domain; acetonitrile might affect antibody activity and tolerance via a non-structural mechanism; dimethylformamide exerted a drastic conformational impact, leading to complete structural disorder, acute activity loss, and the poorest tolerance. Molecular docking results indicated that organic solvents primarily interacted with framework residues via hydrogen bonds, without occupying the core antigen-binding region. This study elucidates the tolerance mechanism of the OTA nanobody in organic solvents, providing a theoretical basis for its application in complex sample detection and the design of solvent-resistant mutants.