Rapid cell-based assay for detection and quantification of active staphylococcal enterotoxin type D

Authors: Rasooly, Reuven; Do, Paula; Hernlem, Bradley - Brad

Submitted to: Journal of Food Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/4/2017
Publication Date: 1/30/2017
Publication URL: http://handle.nal.usda.gov/10113/5678138
Citation: Rasooly, R., Do, P.M., Hernlem, B.J. 2017. Rapid cell-based assay for detection and quantification of active staphylococcal enterotoxin type D. Journal of Food Science. doi: 10.1111/1750-3841.13634.

Interpretive Summary: Food poisoning caused by staphylococcal enterotoxin type D is one of the main causes of food-borne outbreaks. The current method for detection of enterotoxin type D activity uses live monkeys or kittens; however, this expensive procedure raises ethical concerns with regard to the use of experimental animals. In this study we used a combination of a B-cell line to present the toxin to a genetically engineered T cell-line that then produces light. Exposure of the above mixed culture to increasing active staphylococcal enterotoxin type D concentrations produces light intensity in a dose dependent manner.

Technical Abstract: Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, as of the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 hours) quantitative bioluminescence assay, using a genetically engineered T cell Jurkat cell line expressing luciferase under regulation of NFAT response elements (NFAT-RE), in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 105 times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED detection, but reduction in SED activity was shown with heat treatment at 100oC for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED.