Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity

The beta-lactam antibiotics, including penicillins, are the most important antimicrobial substances used for mastitis treatment. Consequently, this is also the most frequently occurring type of antibiotic residues in milk. Today, in addition to the traditional microbial inhibitor tests, rapid and sensitive receptor and immunoassays are used in residue control. Due to the limitations in throughput capacity of these tests, recent applications of automated biosensor technology in food analysis are of great interest. A surface plasmon resonance (SPR)-based biosensor (Biacore) was used to design an inhibition assay to detect beta-lactam antibiotics in milk. A microbial receptor protein with carboxypeptidase activity was used as detection molecule. One advantage of using this receptor protein over antibodies that are more commonly used is that only the active, intact beta-lactam structure is recognized, whereas most antibodies detect both active and inactive forms. In the presence of beta-lactam antibiotics the formation of a stable complex between receptor protein and antibiotic inhibits the enzymatic activity of the protein. The decrease in enzymatic activity was measured using an antibody against the degraded substrate and penicillin G in milk samples was quantitatively determined. The limit of detection of the assay for penicillin G was determined to 2.6 mug kg(-1) for antibiotic-free producer milk, which is below the European maximum residue limit (MRL) of 4 mug kg(-1). The coefficient of variation at 4 mug kg(-1) penicillin G, ranged between 7.3 and 16% on three different days. (C) 2002 Elsevier Science B.V. All rights reserved.