Quantitative analysis of staphylococcus enterotoxin A by differential expression of IFN-γ in splenocyte and CD4⁺ T-cells.
Staphylococcus aureus is an important bacterial pathogen that produces a range of Staphylococcal Enterotoxins (SEs) which cause gastroenteritis and superantigen activation of T cells, the mechanism of which is not well understood. The ability to rapidly detect and quantify SEs is very important in order to learn the causes of staphylococcal outbreaks and to stop similar outbreaks in the future. Enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of several SEs. However, these immunological methods cannot distinguish between active and inactive toxin. It is known that interferon-gamma (IFN-γ) expressed in response to stimulation by SEs contributes significantly to the pathogenesis of S. aureus infection. Nonetheless, the cellular source of IFN-γ is still unclear and the contributions of the specific splenocyte types. In our effort to understand the immunologic response to Staphylococcal Enterotoxin A (SEA) exposure, we studied IFN-γ production in mouse splenocytes. We demonstrated that short term ex vivo exposure of splenocytes or primary naïve CD4+ T-cells to biologically active SEA induces differential expression of IFN-γ mRNA in a time and dose dependent manner and the expression levels reflect the levels of IFN-γ secreted protein. Positive isolated CD4+ T-cells accounted for only 10% of IFN-γ production. We also demonstrate that expression of IFN-γ can be used for rapid quantitative analysis of active SEA with a detection limit of 1 ng/mL.
Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710, USA. reuven.rasooly@ars.usda.gov.
Reuven
R
Rasooly
Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710, USA. bradley.hernlem@ars.usda.gov.
Bradley J
BJ
Hernlem
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004768:Enterotoxins / Q000633:toxicity*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D007371:Interferon-gamma / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D057230:Limit of Detection; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger; D013154:Spleen / Q000187:drug effects / Q000378:metabolism*
D004768:Enterotoxins; D012333:RNA, Messenger; C026066:enterotoxin A, Staphylococcal; D007371:Interferon-gamma