Multiplex lateral flow immunoassay for five antibiotics detection based on gold nanoparticle aggregations

Abstract

A new immunochromatographic assay (ICA) was developed for the simultaneous screening of five antibiotics that can coexist in milk, namely lincomycin (LIN), gentamicin (GEN), kanamycin (KAN), streptomycin (STR), and neomycin (NEO), using five corresponding monoclonal antibodies (mAbs). The five mAbs were conjugated to colloidal gold nanoparticles (AuNPs), forming AuNP-labeled antibodies, which were placed in a microtiter well after freeze-drying; the five antigens were immobilized separately on five test lines to align with their corresponding AuNP-labeled antibodies. Using this method, the cutoff values for the strip test in milk were 25 ng mL⁻¹ for LIN, 25 ng mL⁻¹ for GEN, 50 ng mL⁻¹ for KAN, 50 ng mL⁻¹ for STR, and 100 ng mL⁻¹ for NEO, which are below the maximum residue levels set by the European Union. Based on the strip reader, the multiplex strip could detect the concentrations of LIN, GEN, KAN, STR, and NEO as low as 2.5 ng mL⁻¹, 2.5 ng mL⁻¹, 2.5 ng mL⁻¹, 2.5 ng mL⁻¹, and 5 ng mL⁻¹ in milk, respectively. The accuracy and reproducibility of the assay were acceptable when tested on food samples. In conclusion, our ICA strip is useful for the rapid and high-throughout screening of antibiotics in food and environments on-site.