Epsilon Open Archive

Abstract

The aim of this study was to investigate the applicability of an SPR biosensor in combination with a beta-lactam receptor protein for generic detection of beta-lactam antibiotics in milk. Beta-Lactam antibiotics constitute the group of antimicrobials most commonly used for treatment of bacterial infections in dairy cows. Consequently, they are also the most common type of drug residue found in milk and, as such, causing problems to the dairy industry. The advantage of using a receptor protein, instead of antibodies, is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Three assays based on the same receptor protein, a DD-carboxypeptidase from Actinomadura R39 (R39) were developed using the SPR biosensor Biacore. The first assay was based on the binding of the beta-lactam receptor to a conjugate that was captured on the sensor surface. The assay showed a low detection limit of 1-2 µg/kg for penicillin G in milk, to be compared with the EU maximum residue limit (MRL), which is set to 4 µg/kg. Furthermore, other beta-lactams were detected at or near their respective MRLs. However, there was a high non-specific binding to the sensor surface, varying between different milk samples and batches of conjugate. It was also difficult to obtain a procedure for reproducible synthesis of the conjugate. As an alternative approach, two assays based on the enzymatic activity of the receptor protein were developed. The carboxypeptidase activity of R39 results in hydrolysis of a 3-peptide (Ac-L-Lys-D-Ala-D-Ala) into a 2-peptide (Ac-L-Lys-D-Ala), a reaction which is inhibited in the presence of beta-lactams. Antibodies were developed and used to measure the amount of enzymatic product formed or the amount of remaining enzymatic substrate, respectively. The assays showed low detection limits (1.2 μg/kg and 1.5 μg/kg, respectively) and high precision (CV <5%) for penicillin G in milk and several other beta-lactams were detected at or near their respective MRL. Finally, the two peptide assays were compared with seven commercial tests in screening of producer milk samples. The results showed good agreement and furthermore, the quantitative results obtained were in line with HPLC measurements.