An octuplex lateral flow immunoassay for rapid detection of antibiotic residues, aflatoxin M1 and melamine in milk

Abstract Antibiotic residues, aflatoxin M1 and melamine are three major types of chemical contaminants in milk. However, analytical approach for the simultaneous detection of these chemicals is currently not available. In this study, an octuplex lateral flow immunoassay was developed to address this problem. Specific monoclonal antibodies and receptor were prepared and were bound onto gold nanoparticles to form eight antibody/receptor-gold nanoparticle conjugates as detection probes. Eight corresponding hapten-protein conjugates were synthesized and immobilized on distinct test zones of nitrocellulose membrane as capture antigens. Thus octuplex immune-reactions can then simultaneously occur on a single strip. For qualitative analysis, the result can be visually judged by naked eyes and the cut-off values of quinolones, tetracyclines, β-lactams, sulfonamides, chloramphenicol, streptomycin, aflatoxin M1 and melamine were measured to be 12–80 ng/mL, 16–32 ng/mL, 2–80 ng/mL, 4–80 ng/mL, 1.2 ng/mL, 50 ng/mL, 0.5 ng/mL and 200 ng/mL, respectively. For semi-quantitative analysis, the test lines can be photographed by a built-in camera of smartphone and the line intensities can be analyzed by Image J software. The limits of detection for all the chemicals were far below the regulatory limits set by authorities. Furthermore, the detection time of this method was less than 20 min. Consequently, the developed immunoassay in this study is a promising approach for rapidly screening common chemical contaminants in milk.