Automated Microarray System for the Simultaneous Detection of Antibiotics in Milk

A parallel affinity sensor array (PASA) for the rapid automated analysis of 10 antibiotics in milk is presented, using multianalyte immunoassays with an indirect competitive ELISA format. Microscope glass slides modified with (3-glycidyloxypropyl)trimethoxysilane were used for the preparation of hapten microarrays. Protein conjugates of the haptens were immobilized as spots on disposable chips, which were processed in a flow cell. Monoclonal antibodies against penicillin G, cloxacillin, cephapirin, sulfadiazine, sulfamethazine, streptomycin, gentamicin, neomycin, erythromycin, and tylosin allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with horseradish peroxidase generating enhanced chemiluminescence, which was recorded with a sensitive CCD camera. All liquid handling and sample processing was fully automated, and one analysis was carried out in milk within less than 5 min. The detection limits ranged from 0.12 (cephapirin) to 32 microg/L (neomycin). Penicillin G could be detected at the maximum residue limit (MRL); the detection limits for all other analytes were far below the respective MRLs. The PASA system proved to be the first immunochemical biosensor platform having the potential to test for numerous antibiotics in parallel, such being of considerable interest for the control of milk in the dairy industry.