Use of flow cytometry in an apoptosis assay to determine pH and temperature stability of shiga-like toxin 1.

Shiga toxins and Shiga-like toxins (Stx) are a relatively large group of cytotoxins produced by certain serotypes of Shigella and E. coli (STEC). These toxins are responsible for diarrhea, hemorrhagic colitis and may induce hemolytic uremic syndrome (HUS) with serious consequences in young children. The toxins are proteins made up of 5 small B subunits responsible for binding to an outer membrane ligand on host cells and surround the larger, biologically active A subunit. For Shiga-like toxin 1 (Stx1), the cellular receptor is the carbohydrate globotriose. Stx1was purified from STEC. We utilized induction of apoptosis in the human monocyte cell line THP-1, as a biological endpoint to test the stability of Stx1 activity added to fruit punch at different pH (2-9) and temperatures (4 and 20 degrees C). A flow cytometric method was used to test for early and late apoptotic events based on binding of R-phycoerytherin-labeled annexin V to exposed membrane phosphatidyl serine. Membrane permeability to 7-Amino-actinomycin corresponds with late apoptosis or necrosis. The combination of acid pH and higher storage temperature resulted in greatest degree of toxin inactivation. This approach provides a rapid and high throughput method to determine the functional activity of Stx1, and related toxins in a food matrix.