Multiplex immunochromatographic assay using a universal labeling reactant for determining antibiotic residues in milk.

Background and Aim: In animal husbandry, antibiotics are frequently used as growth promoters, as well as for illness prevention and treatment. They are considered important toxic and allergenic contaminants of food and a serious risk factor for the spread of antibiotic resistance. National and international regulatory authorities have established limits on the permissible residue of antibiotics in food. Immunochromatographic test strips are the most efficient tools for the simple and rapid control of antibiotics for food safety. In these tests, each antibody specific to a particular antibiotic is labeled with a marker, which complicates the manufacturing technology and increases the cost of the test. This study aimed to develop a multiplex immunochromatographic assay (ICA) to determine streptomycin (STR), tetracycline (TC), and chloramphenicol (CAP) residues in milk by indirect labeling of antibiotic-specific antibodies. Materials and Methods: Test strips were composed using 15 μm pore size CNPC nitrocellulose membranes, GFB-R4 separation, and AP045 adsorption pads. The applied reactants include TC and STR conjugates with bovine serum albumin, and CAP-soybean trypsin inhibitor conjugate; anti-TC, anti-STR, and anti-CAP mouse monoclonal antibodies; goat anti-mouse immunoglobulin G (GAMI) conjugated with gold nanoparticles (GNPs) and staphylococcal protein A. Milk samples were collected from cows and goats that had not been injected with any antibiotics. STR and TC/CAP at concentrations of 0.27-600 ng/mL and 0.04-30 ng/mL were added to skim milk, respectively. Milk samples were tested by ICA and calibration curves were constructed to determine the sensitivity of the assay for each antibiotic used. Results: A multiplex ICA of three antibiotic residues in milk was achieved through labeling of immune complexes using a single reagent, GNPs-GAMI. The visual limits of detection (LOD) were 600 ng/mL, 10 ng/mL, and 30 ng/mL for STR, TC, and CAP in cow and goat milk, respectively. Instrumental LODs gave higher sensitivity when analyzed goat milk to STR, TC, and CAP (1.2, 0.05, and 1.3 ng/mL) than cows' milk (7.27, 0.96, and 2.07 ng/mL, respectively). Conclusion: The developed approach for manufacturing multiplex ICA tests for the detection of antibiotic residues in milk does not involve labeling specific antibodies and is implemented using only GNP conjugates with anti-species antibodies.