In vitro cell based assay for activity analysis of staphylococcal enterotoxin A in food

Authors: Rasooly, Reuven; Do, Paula

Submitted to: FEMS Immunology and Medical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/25/2009
Publication Date: 4/27/2009
Citation: Rasooly, R., Do, P.M. 2009. In vitro cell based assay for activity analysis of staphylococcal enterotoxin A in food. FEMS Immunology and Medical Microbiology 56(2009): 172-178.

Interpretive Summary: Staphylococcal entertoxins are a family of protein toxins produced by the bacterium Staphylococcus aureus. Ingestion of toxin can cause severe food poisoning. The “gold standard” assay for measuring the activity of these toxins is a bioassay that uses monkeys or kittens, a method that has low sensitivity and poor reproducibility. This paper describes an improved method for detection of Staphyloccal enterotoxin A (SEA) in food products that have been associated with SEA poisoning outbreaks in the US.Unlike some immunoassays, this new method measures only active toxin. The assay exploits the superantigen activity of SEA that activates large numbers of T cells found in the immune system. This cell-based method is one billion times more sensitive than the monkey or kitten bioassay, and should therefore permit monitoring foods to provide enhanced safety and security, without the use of animals.

Technical Abstract: Staphylococcal enterotoxins (SEs) are a leading cause of food poisoning. They function both as toxins that cause gastroenteritis after ingestion and as superantigens that non-specifically activate large numbers of T cells. Monkey or kitten bioassays were historically developed for analysis of SE activity. To overcome the inherent limitations of such bioassays, this study describes an alternative in vitro method, a splenocyte proliferation assay that measures the superantigen activity of Staphylococcal enterotoxin A (SEA). After incubation of splenocytes with SEA, cell proliferation was measured by labeling the DNA with 5-bromo-2-deoxyuridine (BrdU) and determining BrdU incorporation by spectroscopic measurement. BrdU incorporation is shown to be highly correlated with SEA concentration (R^2=0.99) and can be used to detect 20 pg/ml of SEA, a limit of detection far lower than reported for most ELISAs. Our assay can also distinguish between active toxin and inactive forms of the toxin in foods (milk, chicken and beef). The use of immunomagnetic beads to capture and concentrate the toxin overcame food matrix interference. These results suggest that our in vitro cell-based assay is an advantageous, practical alternative to bioassay for measuring the activity of SEA and possibly other SEs in foods.