Development of an icELISA and immunochromatographic strip for detection of norfloxacin and its analogs in milk

Abstract

We developed a monoclonal antibody (mAb) to detect norfloxacin (NOR) using a novel hapten by introducing an amino group via amido linkage into the piperazine ring of NOR. The sensitivity of an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb showed an improved IC50 value from 1.18 to 0.12 ng/mL and the limit of detection from 0.37 to 0.05 ng/mL by using a heterologous coating (pazufloxacin coupled to ovalbumin). The recovery rate for NOR in milk ranged from 77.3% to 117.8%. Cross-reactivity (CR) experiments showed good CR with nine common fluoroquinolone analogs: pefloxacin, lomfloxacin, ciprofloxacin, enrofloxacin, dafloxacin, sparfloxacin, ofloxacin, marbofloxacin and flumequine. Furthermore, based on this antigen–antibody combination, an immunochromatographic strip with cut-off value of 1 ng/mL was developed. Given their good sensitivity and CR, the developed icELISA and immunochromatographic strip could be useful for detection of NOR and its analogs in milk.