Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/16/2018
Publication Date: 12/15/2018
Publication URL: http://handle.nal.usda.gov/10113/6471043
Citation: Rasooly, R., Do, P.M., He, X., Hernlem, B.J. 2018. Alternative to animal use for detecting biologically active Staphylococcal enterotoxin type A. Toxins. 10(12):540. https://doi.org/10.3390/toxins10120540.
Interpretive Summary: Staphylococcal enterotoxins (SEs) are an important source of food poisoning but methods to detect them either cannot tell inactive toxin from active unless live animals are used. Animal testing is disfavored by ethics and law. We developed for the first time a new method to detect and measure the type A staphylococcal enterotoxin (SEA) using cell lines instead of live animals. These cell lines produce the chemical IL-2 when exposed to SEA. The assay method is highly specific to SEA compared with other SEs (SEB, SED and SEE) and is 10 million times more sensitive than the animal tests. The assay can be used to test food samples, particularly where the SEA is first isolated from the sample.
Technical Abstract: Staphylococcal enterotoxins (SEs) are a food safety concern. Existing methods for biologically active SEs detection rely on the emetic response in live kittens or monkeys. This method suffers from low sensitivity, poor reproducibility and cause ethical concerns regarding the use of experimental animals. The recently passed Lautenberg Chemical Safety Act promotes the development and use of alternatives to animal testing for chemical toxicity methodologies. In this study we utilized the superantigenic effect of SEA and used an ex-vivo bioassay as an alternative to live animal testing. We found that IL-2 secreted by splenocyte can be used for the quantitative detection of SEA in food products. To avoid food matrix interference and attenuation of signal, we employed immunomagnetic beads coated with anti-SEA antibody to separate SEA from spiked food products. This ex-vivo method has enables the detection of 1 ng mL-1 of SEA, that is 107 times more sensitive than the monkey and kitten bioassay. However, this ex-vivo bioassay requires sacrificing of mice. To overcome this limitation, we developed an in vitro cell based assay using CCRF-CEM, a human CD4+ T cell line for the quantitative detection of SEA. Incubation of SEA with CCRF-CEM human T-cells and Raji cells led to quantifiable and dose dependent secretion of IL-2. This novel cell-based assay is highly specific to biologically active SEA, compared with the related SEB, SED, SEE toxin subtypes or heat inactivated SEA which produce no secretion of IL-2. This is the first demonstration of alternative assay that completely eliminating the use of animals for the quantitative detection of biologically active SEA.