Ochratoxin A (OTA) and staphylococcus enterotoxin A (SEA) are highly toxic contaminants and have induced human health problems. They commonly occur in milk and milk products. A competitive fluorescent immunoassay was developed for rapid and simultaneous determination of these toxins in milk samples. The procedure was based on the competitive immunoreactions between antigens in sample and antigen-fluorescent dye conjugates with immobilised antibodies on magnetic nanoparticles (MNPs). Each monoclonal antibody specifically recognises its corresponding toxin (antigen), and there is no cross-reactivity in the assay. First, monoclonal antibodies against OTA and SEA were produced. The activity of the obtained antibodies was determined by fluorescent-linked immunosorbent assay. Then, the monoclonal antibodies were immobilised on MNPs. The amounts of immobilised anti-OTA antibody and anti-SEA antibody were determined to be 20 and 22 μg mL-1, respectively. The antigen-fluorescent dye conjugates OTA-OVA-ATTO620 and SEA-FITC were prepared. The optimal amount of immobilised antibodies for competitive immunoassay was determined. It was found that the linear range of OTA in buffer was larger (0.001-100 ng mL-1) than the linear range of SEA (0.001-20 ng mL-1). The results for simultaneous determination of OTA and SEA in sixfold diluted milk were almost the same in buffer; the linear range for OTA was from 0.005 to 100 ng mL-1 and for SEA from 0.005 to 20 ng mL-1. The detection limit for both OTA and SEA in milk was 0.004 ng mL-1. The developed method took half the time of the individual assays (20 min). The assay was evaluated using spiked milk samples. The influences of somatic cell count, fat, pH and protein concentration in milk on immunoassay were studied. In summary, this developed immunoassay could provide an effective and rapid approach for detecting multi-toxins in milk samples.
Keywords: Fluorescent immunoassay; enterotoxin A; magnetic nanoparticles; milk; ochratoxin A; simultaneous determination.