Simultaneous and rapid detection of five banned antibiotic growth promoters by immunoassay

As a result of increasing concerns over the transfer of resistance between different bacteria and between human and animals, a group of antimicrobial growth promoters including bacitracin, spiramycin, tylosin, virginiamycin and olaquindox have been banned in the EU since 1999. A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) with simple extraction procedures was developed for simultaneous screening of the five banned feed additives in animal feeds. Banned substances were extracted from animal feeds with 70% methanol in water followed by clean-up on OASIS® HLB cartridges. Polyclonal (Pab) antibodies were generated and their specificities assessed by cross-reactivity studies with substantial numbers of antimicrobial agents that are administered to animals via feedingstuffs. The target minimum detectable concentrations (MDC) of 4 mg kg−1 for olaquindox and 1 mg kg−1 for bacitracin, spiramycin, tylosin and virginiamycin were set for the present study. Recoveries at the three levels of the target concentrations (4, 6 and 8 mg kg−1 for olaquindox, 1, 1.5 and 2.0 mg kg−1 for the other four compounds) ranged from 84 to 145% with coefficients of variation less than 18%. The detection capability for virginiamycin, bacitracin, spiramycin, tylosin and olaquindox were 0.2, 0.3, 0.6 and 1.5 mg kg−1, respectively.