Determination of levofloxacin (the Levorotatory Stereoisomer of Ofloxacin) in milk by an indirect enzyme-linked immunosorbent assay

In this work we obtained polyclonal antibodies for levofloxacin. We synthesized conjugates of levofloxacin with cationized BSA for immunization and with ovalbumin for development of an ELISA for the detection of levofloxacin. The method is characterized by a range of detectable concentrations of 0.03 ng/mL to 0.41 ng /mL and the limit of detectable concentrations is 0.01 ng/mL. We tested the 28 fluoroquinolones for cross reactivity and only ofloxacin (145%), marbofloxacin (82%), ofloxacin in its dextrorotatory form (68%), rufloxacin (67%), and garenoxacin (24%) had cross reactivity. The optimized ELISA technique allowed the detection of levofloxacin in milk from 0.33 ng/mL to 3.34 ng/mL. The recoveries were in the range of 89.5–102% with a relative standard deviation of 3%. We tested 45 real samples of milk purchased in local stores; for 5 of them the results were positive (near 1 ng/mL).