Submitted to: PLoS One
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/16/2014
Publication Date: 6/10/2014
Citation: Skinner, C.B., Patfield, S.A., Stanker, L.H., Fratamico, P.M., He, X. 2014. New high-affinity monoclonal antibodies against Shiga toxin 1 facilitate the detection of hybrid Stx1/Stx2 in vivo. PLoS One. 9(6):e99854. DOI:10.1371journal.pone0099854. Interpretive Summary: Shiga toxin 1 is a primary virulence factor for the major bacterial food contaminants Escherichia coli and Shigella dysenteriae. E. coli is responsible for an estimated 100,000 gastrointestinal infections per year in the United States alone. Shigella dysenteriae is a very common pathogen in the developing world, responsible for as many as 90 million infections and 108,000 deaths per year, so there is a great and urgent need for affordable detection of these pathogens. In this study, we report the generation of three new Stx1-specific monoclonal antibodies. All three are capable of neutralizing the toxicity of Stx1 in a Vero cell assay, and two of the three recognize all subtypes of Stx1 (Stx1a, 1c, and 1d). Using a pair of these antibodies, a sandwich ELISA was developed that can detect Stx1 at concentrations as low as 8.7 pg/mL, currently it the most sensitive Stx1-specific immunoassay. Using these new Stx1-specific antibodies and existing Stx2-specific ones, we demonstrate the presence of hybrid Stx1/Stx2 toxin in in vivo samples. These new antibodies and assays will be a boon for detection of Stx1 to ensure a safer food and water supply, and the realization that Stx1/Stx2 hybrid might be present in every E. coli strain that expresses both toxins expands our knowledge of Stx-linked toxicity.
Technical Abstract: Background: Shiga-like toxins (Stxs) are important virulence factors in gastrointestinal infections caused by Shiga toxin-producing Eschericia coli (STEC). Stx1 is almost identical to the Shiga toxin (STx) from Shigella dysenteriae, a very prevalent disease-causing microorganism in the developing world. Developing inexpensive, sensitive methods for detecting Stx1 and STx is a high priority in ensuring the safety of food and water. Methods and Findings: Three novel monoclonal antibodies (mAbs) against Stx1 were produced and characterized. These mAbs recognize the Stx1 B subunit, and do not cross-react with Stx2 or Stx2 subtypes. The optimized Stx1-specific sandwich ELISA using these mAbs has a limit of detection of 8.7 pg/mL, the lowest limit of detection for any Stx1 immunoassay. Two of the three mAbs recognize all three Stx1 subtypes; the third recognizes only Stx1a and Stx1c. All three mAbs can fully neutralize the toxicity of Stx1 in a Vero cell-based assay. Using one of these mAbs and a mAb recognizing only Stx2, we demonstrate the presence of hybrid Stx1/2 in STEC strains that express both Stx1 and Stx2. Conclusions: These new highly sensitive mAbs specific to Stx1 provide new assets for the detection of STEC and Shigella contaminants. Immunoassays with improved sensitivity are demonstrated for the detection of Stx1, and the detection of hybrid Stx1/2 in in vivo samples expands our understanding of the mechanism of the STEC toxicity.