Binding kinetics of monoclonal antibodies and fluoroquinolone derivatives

Abstract

Fluoroquinolones (FQs) are a group of antibiotics which have been extensively used against both Gram-negative and Gram-positive bacteria. Some of FQs antibiotics commonly used in livestock and fishery include norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. However, misuse of these antibiotics may cause drug residues in food products. Therefore, to prevent consumers from getting residual antibiotics in food and the drug resistance of pathogens in humans. As a result, surveillance detection program of these drug residues must be in practice to ensure safety of the consumers in many countries. In general, detections based on immunological method include enzyme-linked immunosorbent assay (ELISA) has been widely used as a screening tool in food safety applications. In this research, sensitivity of four monoclonal antibodies, mAbs against norfloxacin (mAb Nor132 and mAb Nor155), and mAbs against enrofloxacin (mAb Enro44 and Enro48) were quantified by an antigen-captured indirect competitive ELISA. The sensitivity in term of 50% inhibition concentration (IC₅₀) of mAb Nor132, mAb Nor155, mAb Enro44, and mAb Enro48 were found to be 0.1388 µg/ml, 0.0511 µg/ml, 0.2369 µg/ml, and 0.3320 µg/ml respectively. Moreover, the specificity of mAbs were examined in term of % cross-reactivity. The mAb Nor132 and mAb Nor155 are highly specificity for norfloxacin and several drugs in FQs group in range of 21.79-89.34% but mAb Enro44 and mAb Enro48 are highly specificity only enrofloxacin. Then, the kinetic binding (KD) of four mAbs and drugs were studied by surface plasmon resonance (SPR). The result showed that mAb Nor155 bound to norfloxacin and ciprofloxacin better than mAb Nor132 did. As mAb Enro44 bound to enrofloxacin better than mAb Enro48. To investigate In silico binding between FQ drugs and mAbs by using antigen-antibody docking technique, Autodock vina program. The docking results expose significant interactions between the FQ dugs and four mAbs, especially with amino acid residues at the pocket site or nearby the area CDRs of mAbs, the region of mAb recognizes the antigen, with hydrogen bonds. The results indicated that sensitivity and affinity of mAb Nor155 were higher than those of mAb Nor132 as the mAb Enro44 compared to the mAb Enro48. These suggested that mAb Nor155 and mAb Enro44 were suitable for using in the development of norfloxacin and enrofloxacin detection in food products, respectively.