A Rapid and Sensitive Assay for the Detection of Benzylpenicillin (PenG) in Milk

Abstract

Antibiotics, such as benzyl-penicillin (PenG) and cephalosporin, are the most common compounds used in animal therapy. Their massive and illegal use in animal therapy and prophylaxis inevitably causes the presence of traces in foods of animal origin (milk and meat), which creates several problems for human health. With the aim to prevent the negative impact of β-lactam and, in particular, PenG residues present in the milk on customer health, many countries have established maximum residue limits (MRLs). To cope with this problem here, we propose an effective alternative, compared to the analytical methods actually employed, to quantify the presence of penicillin G using the surface plasmon resonance (SPR) method. In particular, the PenG molecule was conjugated to a protein carrier to immunize a rabbit and produce polyclonal antibodies (anti-PenG). The produced antibodies were used as molecular recognition elements for the design of a competitive immune-assay for the detection of PenG by SPR experiments. The detection limit of the developed assay was found to be 8.0 pM, a value much lower than the MRL of the EU regulation limit that is fixed at 12 nM. Thus, our results clearly show that this system could be successfully suitable for the accurate and easy determination of PenG.

Fig 1. PenG molecule and PenG-BSA conjugate.

Penicillin G structure (A) and schematic representation of the PenG-BSA conjugate (B).

Fig 2. SPR binding experiments.

Sensorgram showing the binding of anti-PenG mono-specific antibodies. All measurements were performed in HBS-EP buffer at 25 °C.

Fig 3. SPR-based immunoassay.

Schematic representation of the competitive SPR-based immunoassay for the detection of PenG using a functionalized chip.

Fig 4. SPR competitive immunoassay measurements.

Sensorgram of the competitive immunoassay. All measurements were performed three times in HBS-EP buffer at 25°C.

Fig 5. Calibration curve.

Titration of the SPR-based sensing system with PenG in PBS buffer (black squares) and milk (red circles). RU_max values are plotted versus PenG concentration.