Development of a specific enzyme linked immunosorbent assay (ELISA) for the detection of fluoroquinolone antibiotic residues in chicken liver, prawn and milk

摘要

Extensive utilisation of fluoroquinolones (FQs) in agricultural and aquacultural practices leads to two major food safety issues: 1) the issues regarding the presence of FQs residues in food and 2) the development of FQs resistant bacteria in animals, which may be transferable to humans. This may have an implication to human health, in particular for the treatment of infection. This thesis describes the design and synthesis of novel haptens for (enrofloxacin) ENR, ciprofloxacin and norfloxacin, the production of specific antibodies, and the formatting and characterizing of an indirect competitive Enzyme-Linked ImmunoSorbent Assay (ELISA) for detection of ENR. The design and synthesis of FQs haptens involved the following approaches: 1) synthesising ENR hapten by attaching a tert-butyl linker on a carboxylic group, and 2) synthesising ciprofloxacin and norfloxacin haptens by attaching a 4-bromobutane NHS ester and bromocrotyl NHS ester linkers respectively on the piperazinyl moiety. Highly specific polyclonal antibodies were generated against the ENR-Keyhole Limpet Haemocyanine (KLH) conjugate. The optimized ELISA exhibited higher sensitivity in a homologous assay than a heterologous assay, suggesting the developed antibody was extremely specific to ENR. The ELISA displayed an IC50 value of 11.7 µg L-1 ± 1.7 with a limit of detection (LOD) value of 2.4 µg L-1 ± 0.4. High specificity of the developed assay was evidenced by low cross-reactivity to seven structurally related FQs compounds (danofloxacin, enofloxacin, sarafloxacin, perfloxacin, nalidixic acid, ciprofloxacin and norfloxacin). The effects of surfactants (Tween 20), water miscible organic solvent (methanol, ethanol, acetonitrile, and acetone) and pH conditions (5.5-9.5) were also evaluated. Briefly, Tween 20 affected considerably on colour development, but not the assay sensitivity. Of the solvents tested, up to 5% methanol showed no significant effects on the assay sensitivity. The sample preparation were also optimized for milk, chicken liver and prawn, yielding the recoveries between 64 (± 3) and 125 (± 8)%. This ELISA will be particularly useful for screening ENR residues in animal and marine derived products to improve antibiotic safety in developing countries such as Indonesia.