Abstract:To measure a newly recognized staphylococcal enterotoxin K (SEK), a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was established in this study. The recombinant protein SEK was expressed in BL21 (DE3) pLysS cells and purified by Ni2+-NTA affinity chromatography. Using the protein as an antigen, the monoclonal antibody and polyclonal antibodies were prepared. They were employed for detection and capture of SEK in the DAS-ELISA system that was developed to be capable of detecting SEK in piked skimmed milk, LB medium and minced beef. Subsequently, the ELISA system was applied to determine SEK secretion for 7 sek positive S. aureus isolates with tea polyphenols and Nisin treatment. The results showed that the recombinant protein SEK was successfully expressed and purified with the expected molecular weight of 27.7 ku. The DAS-ELISA for SEK was developed with the optimal anti-SEK monoclonal antibody concentration 2.89 μg/mL, the serum dilution ratio 1:500, the regression equation y=0.2165x+0.1627(R2=0.9993), and the sensitivity 0.1 μg/mL. Using the developed ELISA assay, the recovery rates of SEK in spiked skimmed milk, LB medium and minced beef were more than 97%. Furthermore, both tea polyphenols and Nisin, especially tea polyphenols, inhibited the secretion of SEK at the concentration of MIC.
