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

• Published in: Talanta (Oxford) 2016-03, Vol.149, p.217-224
• Main Authors: Beloglazova, N.V., Shmelin, P.S., Eremin, S.A.
• Format: Article
• Language: English
• Subjects: Animals; Anti-Bacterial Agents - analysis; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - immunology; Antibodies - immunology; Colloidal gold; Fluorescence Polarization Immunoassay; Food Contamination - analysis; Gentamicin; Gentamicins - analysis; Gentamicins - chemistry; Gentamicins - immunology; Gold - chemistry; Lateral-flow test; Metal Nanoparticles - chemistry; Milk; Milk - chemistry; Ovalbumin - chemistry; Quantum dots; Quantum Dots - chemistry
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2015.11.060

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)×109М−1 and (6.0±0.2)×106М−1 for the high- and low-affinity fractions respectively. The assay was characterized with a good sensitivity, the limit of detection being 5μgkg−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, 100μgkg−1, resulting in a 10μgkg−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. [Display omitted] •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.