Lingzi Xiaoli, Dept. of Food Science, Penn State University Kakolie Goswami, Dept. of Food Science, Penn State University Dr. Edward George Dudley, Dept. of Food Science, Penn State University Shiga toxin producing Escherichia coli (STEC) are notorious foodborne pathogens. Upon ingestion of contaminated food, the bacteria establish themselves in the colon and produce Shiga toxin (Stx) which translocates through the endothelial membrane and reaches the target cells (1–3).
Disease symptoms first present as a mild diarrhea that can progress to bloody diarrhea and further lead to hemolytic uremic syndrome or hemorrhagic colitis. There are two immunologically distinct Shiga toxin, designated Shiga toxin 1 (Stx1) and 2 (Stx2). Studies have shown that Stx2 is more potent than Stx1 (4). While Stx can be detected by commercially available kits, these methods are not quantitative. Additionally, the only commercially available anti-Stx antibodies are mouse monoclonals, so designing an enzyme-linked immunosorbent assay (ELISA) requires one to generate antibodies from another species to use for capture or detection.
We were interested in designing an ELISA using only commercially available components, and below we describe how we accomplished this using ceramide trihexoside (also known as globotriaosylceramide, Gb3, and CTH) obtained from Matreya.
* Matreya is very pleased to have this article from professor E. G. Dudley at The Pennsylvania State University. We thank Dr. Dudley and his group for their collaboration. Shiga toxin 2 binds to the glycolipid globotriaosylceramide (Gb3) on the kidney epithelium. In our study, three different types of CTH were individually used to coat a polystyrene 96 well plate. Both the top (non-hydroxy acyl) and bottom (hydroxy acyl) spot of CTH as well as the semisynthetic lyso-CTH were compared, and we concluded that the bottom spot CTH was most sensitive for Stx quantification. We also investigated the efficacy of different solvents to dissolve the CTH bottom spot. Hot methanol (at 55oC), room temperature methanol and chloroform: methanol (2:1) were tested. CTH bottom spot dissolved in the chloroform:methanol (2:1) gave the highest signal. We also tested a combination of cholesterol, lecithin and CTH (bottom spot), however the bottom spot alone showed highest