Characterization\nof a Novel Monoclonal Antibody with\nHigh Affinity and Specificity against Aflatoxins: A Discovery from\nRosetta Antibody-Ligand Computational Simulation

Aflatoxin B1 (AFB<sub>1</sub>) accumulates in crops,\nwhere it poses\na threat to human health. To detect AFB<sub>1</sub>, anti-AFB<sub>1</sub> monoclonal antibodies have been developed and are widely\nused. While the sensitivity and specificity of these antibodies have\nbeen extensively studied, information regarding the atomic-level docking\nof AFB<sub>1</sub> (and its derivatives) with these antibodies is\nlimited. Such information is crucial for understanding the key interactions\nthat are required for high affinity and specificity in aflatoxin binding.\nFirst, a 3D comparative model of anti-AFB<sub>1</sub> antibody (Ab-4B5G6)\nwas predicted from the sequence using RosettaAntibody. We then utilized\nRosettaLigand to dock AFB<sub>1</sub> onto ten homology models, producing\na total of 10,000 binding modes. Interestingly, the best-scoring mode\npredicted strong interactions involving four sites within the heavy\nchain: ALA33, ASN52, HIS95, and TRP99. Importantly, these strong binding\ninteractions exclusively involve the variable domain of the heavy\nchain. The best-scoring mode with AFB<sub>1</sub> was also obtained\nthrough AF multimer combined with RosettaLigand, and two interactions\nat TRP and HIS were consistent with those found by Rosetta antibody-ligand\ncomputational simulation. The role of tryptophan in π interactions\nin antibodies was confirmed through mutation experiments, and the\nresulting mutant (W99A) exhibited <i>a</i> >1000-fold\nreduction\nin binding affinity for AFB<sub>1</sub> and analogs, indicating the\neffect of tryptophan on the stability of CDR-H3 region. Additionally,\nwe evaluated the binding of two glycolic acid-derived molecular derivatives\n(with impaired hydrogen bonding potential), and these derivatives\n(AFB<sub>2</sub>-GA and AFG<sub>2</sub>-GA) demonstrated a very weak\nbinding affinity for Ab-4B5G6. The heavy chain was successfully isolated,\nand its sensitivity and specificity were consistent with those of\nthe intact antibody. The homology models of variable heavy (VH) single-domain\nantibodies were established by RosettaAntibody, and the docking analysis\nrevealed the same residues, including Ala, His, and Trp. Compared\nto the potential binding mode of fragment variable (FV) region, the\nresults from a model of VH indicated that there are seven models involved\nin hydrophobic interaction with TYR32, which is usually referred to\nas polar amino acid and has both hydrophobic and hydrophilic features\ndepending on the circumstances. Our work encompasses the entire process\nof Rosetta antibody-ligand computational simulation, highlighting\nthe significance of variable heavy domain structural design in enhancing\nmolecular interactions.