RESUMEN
In this study, an anti-amoxicillin single chain variable fragment (ScFv) antibody was evolved by directional mutagenesis of a contact amino acid residue based on the analysis of virtual mutation. Comparison with its parental ScFv, the mutant showed highly improved affinity for 11 penicillins with up to 6-folds increased sensitivity. Then, its recognition mechanisms for the 11 drugs were studied by using molecular docking. Results showed that the mutant-penicillins intermolecular forces increased and the total binding energies decreased dramatically, which were responsible for the improvement of antibody sensitivity. The ScFv mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for determination of the 11 drugs in milk. The limits of detection were in the range of 0.2-3.0 ng/mL, the crossreactivities were in the range of 31%-132%, and the recoveries from standards fortified blank milk were in the range of 65.7%-92.4%. This is the first study reporting the directional evolution of a ScFv antibody based on virtual mutation and the use of ScFv antibody for determination of penicillins in foods of animal origin.
Asunto(s)
Amoxicilina/análisis , Evolución Molecular Dirigida/métodos , Análisis de los Alimentos/métodos , Leche/química , Mutación Missense , Anticuerpos de Cadena Única , Sustitución de Aminoácidos , Amoxicilina/química , Animales , Bovinos , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genéticaRESUMEN
A recombinant antisarafloxacin ScFv antibody was produced by direct transformation of its gene into Rosetta-gami(DE3) for expression, and then its recognition mechanisms for 12 fluoroquinolones were studied using the molecular docking method. On the basis of the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Tyr99 to His. The ScFv mutant showed highly increased affinity for the 12 drugs with up to sevenfold improved sensitivity. Finally, the mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for determination of the 12 drugs in milk. The limits of detection were in the range of 0.3-8.0 ng/mL; the ties were in the range of 5-106%, and the recoveries from the standard fortified blank milk were in the range of 62.0-89.3%. This is the first study reporting the evolution of an ScFv antibody using a directional mutagenesis strategy based on virtual mutation.
