A sensitive and semi-quantitative method for determination of multi-drug residues in animal body fluids using multiplex dipstick immunoassay

• A multiplex dipstick immunoassay was developed to detect 3 families of antibiotic residues in milk, urine, and serum.
• Samples did not require pretreatment and could be directly analyzed within 10 min.
• The method showed high sensitivity and specificity with false positive ≤5%.

The objective of this research was to develop a multiplex dipstick immunoassay method for the simultaneous determination of multi-veterinary drug residues, such as β-agonists, sulfonamides, and tetracyclines in milk, urine, and serum. The multiplex dipstick assay format was based on an indirect competitive approach: Three test lines (different antigens) and one control line (goat anti-mouse IgG) were located on the strip membrane. Labeled antibodies were freeze-dried in microwells. Samples did not require pretreatment and could be directly analyzed within 10 min. Threshold levels in different sample matrices were visually estimated at 0.3–0.45 ng mL−1 for clenbuterol; 3–4 ng mL−1 for sulfadiazine; and 4.5–6 ng mL−1 for tetracycline, respectively. The linear relationship between the concentrations of veterinary drug residues and the Au nanoparticles plasmon absorbance allowed quantitative determination of these veterinary drug residues. The recoveries of clenbuterol, sulfadiazine and tetracycline in spiked samples ranged from 78.4% to 112.6%, and the relative standard deviations were below 11.2%. Analysis of animal samples suggested that the proposed multiplex dipstick assay method was consistent with the LC-MS/MS method. The percentage of false results was less than or equal to 5%. Thus, the proposed multiplex dipstick assay is inexpensive, easy-to-use, and suitable for the purposes of rapid and comprehensive screening of 3 families of β-agonists, sulfonamides and tetracyclines including 26 drugs in animal body fluids.

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