Indirect competitive ELISA and colloidal gold-based immunochromatographic strip for amantadine detection in animal-derived foods

In this research, an indirect competitive enzyme-linked immunosorbent assay (ELISA) and colloidal gold-based immunochromatographic (GICG) strip were developed for the detection of the antiviral drug amantadine (AM) in animal-derived foods. Under the optimized conditions of coating antigen (AM-BSA) content of 0.01 μg per well, antibody concentration of 145.3 ng mL⁻¹, 0.5% defatted milk powder solution as the blocking solution, and phosphate buffer solution (0.01 mol L⁻¹, pH 7.4) as the diluent, the ELISA displayed a linear range of 1.0–80.0 μg L⁻¹ for AM, with good sensitivity (IC₅₀: 4.2 μg L⁻¹) and detection limit (LOD: 1.0 μg L⁻¹). The recoveries of AM in selected animal-derived samples ranged from 85.2% to 115.6%. Under the optimized conditions for the GICG with a whole antigen and second antibody dilution of 40- and 60-fold, respectively, pH of the probe coupling buffer of 8 and concentration of gold-labeled antibody of 5 μg mL⁻¹, the visual LOD of the GICG was 10 μg L⁻¹ and the detection was completed within 10 min with a relatively simple pretreatment of the food samples. The developed ELISA and GICG strip also showed high specificity with low cross-reactivity toward AM analogues and other drugs. These results indicate that the developed ELISA and GICG strip offer the accurate, sensitive, specific, and fast detection of AM in animal-derived food samples.