Biosensor analysis of beta-lactams in milk using the carboxypeptidase activity of a bacterial penicillin binding protein.
The applicability of a beta-lactam receptor protein for detection of beta-lactam antibiotics in milk using surface plasmon resonance (SPR) biosensor technology was investigated. The advantage of using a receptor protein instead of antibodies for detection of beta-lactams is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Two assays based on the enzymatic activity of the DD-carboxypeptidase from Actinomadura R39 were developed, using a Biacore SPR biosensor. The carboxypeptidase converts a tri-peptide into a di-peptide, a reaction which is inhibited in the presence of beta-lactams. Polyclonal antibodies against the 2 peptides were developed and used to measure the amount of enzymatic product formed (di-peptide assay) or the amount of remaining enzymatic substrate (tri-peptide assay), respectively. The 2 assays showed similar performances with respect to detection limits (1.2 and 1.5 microg/kg, respectively) and precision (coefficient of variation <5%) for penicillin G in milk. Several other beta-lactams were detected at or near their respective maximum residue limit. Furthermore, the 2 peptide assays were evaluated against 5 commercial kit tests in the screening of 195 producer milk samples. The biosensor assays showed 0% false-negative and 27% false-positive results, whereas the figures were 0% false-negative and 27-53% false-positive results for other screening tests investigated.
Sternesjö
Ase
A
Swedish University of Agricultural Sciences, Department of Food Science, PO Box 7051, 750 07 Uppsala, Sweden. ase.sternesjo@lmv.slu.se
Gustavsson
Eva
E
D000818:Animals; D000918:Antibody Specificity; D015374:Biosensing Techniques / Q000379:methods*; D002268:Carboxypeptidases / Q000032:analysis* / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods; D005188:False Negative Reactions; D005504:Food Analysis / Q000379:methods*; D008892:Milk / Q000378:metabolism*; D046915:Penicillin-Binding Proteins / Q000032:analysis*; D010455:Peptides / Q000737:chemistry; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance; D047090:beta-Lactams / Q000032:analysis*
D046915:Penicillin-Binding Proteins; D010455:Peptides; D047090:beta-Lactams; D002268:Carboxypeptidases