Site-directed mutations of anti-amantadine scFv antibody by molecular dynamics simulation: prediction and validation

A recombinant single-chain variable fragment (scFv) antibody was produced from a hybridoma cell strain secreting the monoclonal antibody for amantadine (AMD), and then its recognition mechanisms for AMD were studied using the molecular docking and molecular dynamics. Complex dockings revealed that three regions are involved in antibody recognition; framework 2 of the VL chain (LFR2) GLU40 and TYR42, complementarity-determining region of the VL chain (LCDR3) TYR116, and framework 2 of the VH chain (HFR2) HIS40 and TRP52 were the key amino acid residues. The results of molecular dynamics show that the most important amino acid residues in the interaction between AMD and scFv are HIS40 and TYR116. On the basis of the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of amino acid residue GLY107 to PHE. Indirect competitive ELISA (icELISA) results indicated that the scFv mutant had highly increased affinity for AMD with up to 3.9-fold improved sensitivity. Thus, the scFv antibody can be applied for mechanistic studies of intermolecular interactions, and our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-AMD antibody design and preparation in future.