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Development of Fluorescence Polarisation and Enzyme-Linked Immunosorbent Assays for Danofloxacin Detection In Milk

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%.