Advances in Food Technology and Nutritional Sciences

Aim: This study develops a quantitative ELISA for measuring Shiga toxin 2 (Stx2) produced by Shiga toxin (Stx)-producing Escherichia coli (STEC), including foodborne pathogen E. coli O157:H7 by all commercially available reagents. Background: Most foodborne outbreak strains of STEC produce Stx2a, Stx2c or both, which are more frequently associated with human clinical cases, leading to severe gastrointestinal diseases or even death. However, no simple and cheap assays or kits are available for quantitative detection of Stx2a and Stx2c. Therefore, an easy and affordable quantitative method for Stx2 is needed for its pathogenesis study. Results: We successfully developed a sensitive and specific receptor-based ELISA by using all commercial available agents. Hydroxyl acyl ceramide trihexoside, an analogue of Stx2 receptor globotriaosylceramide (Gb3), was used for antigen capture, and several critical steps were identified that must be adhered to ensure repeatability. No cross reactivity was observed with Stx1, and linear curves could be constructed using either purified Stx2a or a bacterial lysate from an Stx2a-producing E. coli O157:H7 strain. We applied this method to quantify Stx2 production by a collection of E. coli O157:H7 strains, indicating it can be extended to qualitatively evaluate Stx2c, and providing evidence that toxin production does not necessarily correlate with strain phylogeny. Conclusion: Our R-ELISA provides a reliable way to quantify Stx2a using commercially available components, and it can also be used for detecting Stx2c. This cost-effective ELISA can be easily performed, suggesting it will be a useful tool for studying pathogenesis of STEC.