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

Area of Science:

• Food Safety and Analytical Chemistry
• Immunotechnology and Biosensing
• Veterinary Pharmacology and Residue Analysis

Background:

• Antibiotics in animal husbandry pose risks as food contaminants and drivers of antibiotic resistance.
• Regulatory bodies set strict limits for antibiotic residues in food products.
• Current immunochromatographic assays (ICAs) for antibiotic detection are complex and costly due to individual antibody labeling.

Purpose of the Study:

• To develop a cost-effective multiplex immunochromatographic assay (ICA) for simultaneous detection of streptomycin (STR), tetracycline (TC), and chloramphenicol (CAP) in milk.
• To simplify the manufacturing process of antibiotic testing strips by using indirect labeling of antibodies.

Main Methods:

• Constructed test strips using nitrocellulose membranes and various absorption/separation pads.
• Utilized specific antibiotic-conjugates (e.g., TC-BSA, STR-BSA, CAP-STI) and monoclonal antibodies (anti-TC, anti-STR, anti-CAP).
• Employed a single gold nanoparticle (GNP)-conjugated reagent (goat anti-mouse IgG) for indirect labeling of immune complexes.

Main Results:

• Successfully developed a multiplex ICA capable of detecting three antibiotic residues in milk.
• Achieved visual limits of detection (LODs) of 600 ng/mL for STR, 10 ng/mL for TC, and 30 ng/mL for CAP.
• Instrumental analysis demonstrated higher sensitivity, with LODs as low as 1.2 ng/mL (STR), 0.05 ng/mL (TC), and 1.3 ng/mL (CAP) in goat milk.

Conclusions:

• The developed multiplex ICA offers a simplified and potentially more economical approach for detecting multiple antibiotic residues in milk.
• The indirect labeling strategy using a single GNP-conjugated reagent significantly streamlines the manufacturing of ICA test strips.
• This method provides a valuable tool for rapid and efficient food safety monitoring of antibiotic contamination.