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United States Department of Agriculture

Agricultural Research Service

Research Project: Technologies for Detecting and Determining the Bioavailability of Bacterial Toxins

Location: Foodborne Toxin Detection and Prevention

Title: A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by escherichia coli in human and environmental samples

Authors
item He, Xiaohua
item Patfield, Stephanie
item Hnasko, Robert
item Rasooly, Reuven
item Mandrell, Robeert -

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 28, 2013
Publication Date: October 16, 2013
Citation: He, X., Patfield, S.A., Hnasko, R.M., Rasooly, R., Mandrell, R.E. 2013. A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by escherichia coli in human and environmental samples. PLoS One. 8(10):e76368. DOI:10.1371/journal.pone.0076368.

Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) are a group of bacteria responsible for many outbreaks of human gastrointestinal diseases. Shiga toxin 2 (Stx2) was found to play the most important role in the clinical outcome of STEC infection. Although there are seven subtypes of Stx2 and all of them have been known to be associated with human illnesses, there is no single assay available for detection of all these subtypes. In this study, we developed a polyclonal antibody and a simple sandwich ELISA using this antibody for sensitive detection of Stx2. To our knowledge, this is the first assay that is capable of detecting all subtypes of Stx2 produced by E. coli from human and environmental samples. It will be useful for rapid diagnosis of Stx2 contamination in biological samples, thus reducing the risk of STEC outbreak.

Technical Abstract: The increase of outbreaks and illnesses linked to Shiga toxin-producing Escherichia coli (STEC) has necessitated the development of effective detection methods for these pathogens in various matrices. The best way to determine if a bacterial strain is a STEC is to examine the production of Shiga toxin (Stx). Here we describe the preparation of a polyclonal antibody using the recombinant toxoid, Stx2E167Q, as an immunogen. This polyclonal antibody binds specifically to the A-subunit of Stx2 and is capable of neutralizing Stx2-mediated cytotoxicity in Vero cells. A simple, rapid, and sensitive ELISA capable of detecting all subtypes of Stx2 was developed using this polyclonal antibody with a limit of detection between 10 and 100 pg/mL for Stx2a. When applied to 36 cultural supernatants of bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all samples that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity. To our knowledge, this is the first ELISA that is able to detect all subtypes of Stx2 in bacterial cultural supernatants. This assay will enable any competent laboratory to identify and characterize Stx2-positive O157 and non-O157 STEC and provide rapid diagnosis and improved patient care.

Last Modified: 9/23/2014
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