Development of Fluorescence Polarisation and Enzyme-Linked Immunosorbent Assays for Danofloxacin Detection In MilkAuthor(s): I. A. Shanin, E. A. Zvereva, A. V. Zherdev, S. A. Eremin and B. B. Dzantiev
Two selective immunotechniques based on heterologous approach were proposed and chacterized to detect danofloxacin, a priority veterinary contaminant, in milk. The first technique is fluorescence polarisation immunoassay (PFIA), where a danofloxacin tracer was synthesised with 4-aminomethyl fluorescein, with polyclonal antibodies obtained for a danofloxacin conjugate with cationised bovine serum albumin. The developed system has a detection limit for danofloxacin equal to 13 ng/mL and does not show any cross-reactivity with other 24 tested fluoroquinolones. The second assay format is enzymelinked immunosorbent assay (ELISA) for danofloxacin that is based on the application of the same antibodies and a heterologous solid-phase clinafloxacin conjugate with ovalbumin. The limit of danofloxacin detection for the given ELISA is 0.5 ng/mL, and its strong selectivity to danofloxacin has been also confirmed. The possibility of quantitative control of danofloxacin content in milk has been shown for the developed ELISA technique; the opening percentage was in the range from 83 to 130%.