English

A biospecific interaction assay (BIA) for the detection of residues of ß-lactam antibiotics in milk was developed using a surface plasmon resonance (SPR) biosensor. Penicillin-binding proteins (PBP), which are enzymes involved in the final stages of murein biosynthesis and represent the lethal target of ß-lactam antibiotics, were used as biomolecular recognition partners for penicillins and cephalosporins. PBP were mixed with samples, incubated and then labelled with an antigen-tagged ampicillin-derivative prior to SPR analysis with antibodies against the antigen-tag immobilised on the sensor chip. In order to detect residues of ß-lactam antibiotics at concentrations corresponding to maximum residue limits (MRL) set by the European Union, a suitable PBP, an antigen-tagged ampicillin-derivative and an antibody against the antigen had to be found. PBP of Streptococcus thermophilus and Bacillus stearothermophilus were partly purified by ampicillin affinity chromatography and a soluble derivative of PBP 2x of Streptococcus pneumoniae was expressed as a recombinant protein in Escherichia coli. This soluble PBP 2x-derivative, called PBP 2x, had higher affinities towards different ß-lactam antibiotics than the other studied PBP and was chosen for construction of the ß-lactam-specific BIA. Ampicillin (AMPI) was labelled with derivatives of digoxigenin (DIG), fluorescein (FLU) or 2,4 dinitrophenol (DNP). DIG, FLU and DNP were coupled through their activated N-hydroxysuccinimide esters to the amino group of AMPI, yielding DIG labelled AMPI (DIG AMPI), FLU labelled AMPI (FLU-AMPI) and DNP labelled AMPI (DNP-AMPI), respectively. DIG AMPI, FLU-AMPI and DNP AMPI were examined as antigen-tagged ampicillin-derivatives. Benzylpenicillin, ampicillin, amoxicillin, cloxacillin, cephalexin and cefoperazone could be detected in commingled raw milk at concentrations below their respective MRL using PBP 2x, DIG-AMPI and the monoclonal antibody against DIG for SPR analysis of milk samples. The SPR analysis of 20 raw milk samples from individual cows confirmed that matrix interferences are a critical point in BIAs as non-specific binding of matrix components to the sensor chip elevated the detection limit dramatically. Matrix interferences could be reduced to a lower and a more constant level by a heat treatment step and the addition of carboxymethylated dextran to the samples. With this additional sample preparation, individual samples containing benzylpenicillin at the MRL were identified as positive.

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