A new electrochemical enzyme-linked immunosorbent assay for the screening of macrolide antibiotic residues in bovine meat

A new sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) f or the detection of two macrolides (erythromycin and tylosin) in bovine mus cle was developed, using the mouse monoclonal antibodies anti-erythromycin and anti-tylosin. The competitive indirect assay was performed using an ery thromycin (or tylosin)-BSA conjugate as a coating molecule; after competiti on between free and coated analytes for the antibodies, the activity of the horseradish peroxidase-labelled antiglobulins was measured electrochemical ly using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate. The detection l imit of the assay was 0.4 ng ml(-1) for erythromycin and 4.0 ng ml(-1) for tylosin, while the sensitivity (25% inhibition concentration) was 1.4 ng ml (-1) for erythromycin and 13.0 ng ml(-1) for tylosin. The specificity of th e assay was assessed by studying the cross-reactivity of various macrolides other than erythromycin and tylosin. The results indicate that the monoclo nal antibodies anti-erythromycin and anti-tylosin can readily distinguish t he target compound from other macrolides, with the exception of roxithromyc in, a semisynthetic macrolide antibiotic derived from erythromycin. Fortifi ed and real samples were analysed by the developed ELISA method and results confirmed by micro-LC-MS-MS using an atmospheric pressure ionisation (API) source and an ionspray (IS) interface. The latter provides unequivocal ide ntification and quantification of the analytes at the level of interest. Th e ELISA assay showed precision (RSD) values ranging from 6.3 to 11.4% for e rythromycin and from 7.5 to 12.6% for tylosin; the accuracy (relative error , RE) ranged from -16.0 to -9.8% and from -9.5 to 8.0% for erythromycin and tylosin, respectively. All results obtained demonstrate that the electroch emical ELISA is a suitable method for a sensitive, simple, rapid and reliab le screening of the two macrolides in animal tissues.