Sensitive immunochemical approaches for quantitative (FPIA) and qualitative (lateral flow tests) determination of gentamicin in milk

• A sensitive FPIA for the determination of gentamicin in milk was developed.
• Colloidal gold-based lateral flow immunoassay for gentamicin detection was designed.
• Quantum dots were used as label in lateral flow test against gentamicin.
• The techniques were validated using naturally-contaminated and spiked milk samples.

Three kinds of immunoassays for the determination of gentamicin in milk samples were developed and validated. First, a fast and easily-performed fluorescence polarization immunoassay was used for characterization of the employed polyclonal antibody. The calculated Kaff were (1.9±0.4)×109 М−1 and (6.0±0.2)×106 М−1 for the high- and low-affinity fractions respectively. The assay was characterized with a good sensitivity, the limit of detection being 5 μg kg−1. Two different kinds of detection labels, i.e. colloidal gold (CG) and quantum dots (QDs), were evaluated for use in lateral-flow format with respect to rapid visual on-site testing. The cut-off levels for both qualitative formats were selected based on the maximum level for gentamicin in milk established by the European Commission, 100 μg kg−1, resulting in a 10 μg kg−1 cut-off considering sample dilution. The intra-laboratory validation was performed with sterilized milk samples artificially spiked with gentamicin at concentrations less than, equal to, and greater than the cut-off level. It was shown that milk products could be analyzed without any sample preparation, except for dilution with the buffer solution. The rates of false-positive and false-negative results were below 5% for both labels. The different developed immunoassays were tested towards gentamicin determination in artificially-spiked and naturally contaminated milk samples.

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