|RUTH, LEAH - Abraxis, Llc|
|FERNANDO, RUBIO - Abraxis, Llc|
Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/17/2018
Publication Date: 10/23/2018
Citation: Armstrong, C.M., Ruth, L., Capobianco Jr, J.A., Strobaugh Jr, T.P., Fernando, R., Gehring, A.G. 2018. Detection of shiga toxin 2 produced by Escherichia coli in foods using AlphaLISA. Toxins. 10(11):422. https://doi.org/10.3390/toxins10110422.
Interpretive Summary: Food poisoning due to Shiga toxin-producing Escherichia coli (STEC) is a consistent concern in the United States because of the high costs associated with STEC illnesses; from unscheduled work absence to hospitalization and possibly death. The production of Shiga toxin (Stx) is known to be essential for the development of some of the more severe medical conditions resulting from STEC infections, including hemolytic uremic syndrome. Because of the serious implications surrounding the production of Stx, the Food Safety and Inspection Service has undertaken routine screening of meat samples for the presence of the gene that produces the toxin as a method of identifying the presence/absence of STEC in the food supply chain. Here we have developed a bead-based immunoassay (known as an AlphaLISA) for the detection of Stx and tested it in both Romaine lettuce and ground beef. This assay was found to meet the current industrial standard for Stx detection (limits of 0.5 parts-per-billion) yet has several advantages over these approaches including a more rapid testing time and a larger dynamic range. In addition, it is easily amendable to automation and high-throughput screening, making it a desirable alternative for Stx detection in food.
Technical Abstract: Amplified Luminescent Proximity Homogenous Assay-linked Immunosorbent Assay (AlphaLISA) is comprised of a bead-based immunoassay that is used for small molecule detection. In this study, a novel AlphaLISA was developed and optimized for the detection of Shiga-toxin 2 (Stx2). Efficacy and sensitivity trials showed the AlphaLISA could detect =0.5 ng/mL of purified Stx2, which was comparable to the industry standard enzyme-linked immunosorbent assay (ELISA) tests for Stx2 detection. In addition, evaluation of Shiga toxin-producing Escherichia coli (STEC)-inoculated Romaine lettuce and ground beef samples demonstrated that both the AlphaLISA and the ELISA were able to discern uninoculated samples from 1X and 10X diluted samples containing ~10 CFU/mL of STEC enriched in modified tryptic soy broth with mitomycin C for 16 h. Overall, the increased signal-to-noise ratios indicated a more robust signal was produced by the AlphaLISA compared to the ELISA and the delineation of higher toxin concentrations without the need for sample dilution implied a greater dynamic range for the AlphaLISA. Implementation of the newly developed AlphaLISA will allow for more rapid analysis for Stx2 with less manual manipulation thus, improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 parts-per-billion.