Simultaneous detection of sulfamethazine, streptomycin, and tylosin in milk by microplate-array based SMM–FIA

This paper presents an approach to simultaneously detect sulfamethazine, streptomycin, and tylosin in milk by indirect competitive multianalyte Fluorescence immunoassay (FIA). Microscope glass slides modified with agarose were used for the preparation of small molecule microarrays (SMMs). Bovine serum albumin (BSA) conjugates of the haptens were immobilized on glass slides. The system consists of four glass slides containing 96 wells formed by an enclosing hydrophobic mask, which precisely matches a standard microplate. All liquid handling and sample processing were fully automated as 96-wells ELISA format. Monoclonal antibodies against sulfamethazine, streptomycin, and tylosin allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with Cy5 generating fluorescence, which was scanned with chip scanner. The detection limits for three analytes were 3.26 ng/ml (sulfamethazine), 2.01 ng/ml (streptomycin), and 6.37 ng/ml (tylosin), being far below the respective MRLs. The system proved to be the first SMM–FIA platform having the potential to test for numerous antibiotics in parallel, such being of considerable interest for the control of safety in the food industry.