title: In vitro cell-based assay for activity analysis of staphylococcal enterotoxin A in food.
abstract: Staphylococcal enterotoxins (SEs) are a leading cause of food poisoning and have two separate biological activities; it causes gastroenteritis and functions as a superantigen that activates large numbers of T cells. In vivo monkey or kitten bioassays were developed for analysis of SEs emetic activity. To overcome the inherent limitations of such bioassays, this study describes an in vitro splenocyte proliferation assay based on SEs superantigen activity as an alternative method for measuring the activity of staphylococcal enterotoxin A (SEA). After incubation of splenocytes with SEA, cell proliferation was measured by labeling the proliferating cells' DNA with bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) and quantifying the incorporated BrdU by immunohistochemistry. BrdU labeling is shown to be highly correlated with SEA concentration (R(2)=0.99) and can detect 20 pg mL(-1) of SEA, which is far more sensitive than most enzyme-linked immunosorbent assays. Our assay can also distinguish between active toxin and inactive forms of the toxin in milk. By applying immunomagnetic beads that capture and concentrate the toxin, our assay was able to overcome matrix interference. These results suggest that our in vitro cell-based assay is an advantageous practical alternative to the in vivo monkey or kitten bioassays for measuring SEA and possibly other SEs activity in food.
authors.affiliation: US Department of Agriculture, Western Regional Research Center, Agricultural Research Service, Albany, CA 94710, USA. reuven.rasooly@ars.usda.gov
authors.forename: Reuven
authors.initials: R
authors.lastname: Rasooly
authors.forename: Paula M
authors.initials: PM
authors.lastname: Do
mesh_terms: D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004768:Enterotoxins / Q000032:analysis* / Q000633:toxicity*; D005260:Female; D005504:Food Analysis; D005516:Food Microbiology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects*; D051379:Mice; D008810:Mice, Inbred C57BL; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology; D013194:Staining and Labeling / Q000379:methods
chemical_list: D004768:Enterotoxins; C026066:enterotoxin A, Staphylococcal; D004247:DNA; D001973:Bromodeoxyuridine