Submitted to: PLoS One
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/31/2013
Publication Date: 9/17/2013
Citation: Skinner, C.B., Patfield, S.A., Stanker, L.H., He, X. 2013. Development of monoclonal antibodies and immunoassays for sensitive and specific detection of Shiga Toxin Stx2f. PLoS One. 8(9):e76563 DOI: 10.1371/journal/pone.0076563. Interpretive Summary: A major bacterial food contaminant, E. coli expressing Shiga-like toxin (STEC) are responsible for several recent deadly outbreaks. Shiga-like toxin 2 is a critical virulence factor in these STEC outbreaks and is associated with potentially life-threatening Hemolyic-Uremic Syndrome (HUS). There are many subtypes of Stx2 (Stx2a through Stx2g), several of which (Stx2e and Stx2f) are difficult to detect and purify, and are therefore uncharacterized. Stx2f is the most diverse of these Stx2 subtypes. In this study, we report the generation of four monoclonal antibodies that are used for sensitive and specific detection of the Stx2f subtype. Using a pair of these antibodies, a sandwich ELISA was developed that can detect Stx2f at concentrations as low as 123 pg/mL. Three of the four antibodies are partially neutralizing (mAbs Stx2f-1, 2, and 4) in a Vero cell toxicity assay. Since Stx2f is so difficult to detect by conventional means, Stx2f may be present in many more isolates of EHEC and STEC than we realize. To facilitate identification of Stx2f-expressing strains, we developed a colony immunoblot assay that can detect Stx2f specifically from petri dish mixed cultures. These new antibodies and assays will greatly simply detection and characterization of Stx2f- expressing STEC, and ensure a safer food supply.
Technical Abstract: Background: Shiga-like toxin 2 (Stx2) is a major virulence factor in gastrointestinal diseases caused by Eschericia coli. Although Stx2a (prototypical Stx2) is well-studied, all seven subtypes of Stx2 have been associated with disease in mammals. Several subtypes of Stx2, including Stx2f, are difficult to detect immunologically. Methods and Findings: Four novel monoclonal antibodies (mAbs) against the Stx2f subtype were produced and characterized. These mAbs react exclusively to the Stx2f A subunit, and do not cross-react with other subtypes of Stx2. A Stx2f-specific sandwich ELISA was established and a limit of detection of 0.123 ng/mL was obtained using one pair of the mAbs. The receptor preference of Stx2f was confirmed using this sandwich ELISA. Three out of four mAbs can partially neutralize the toxicity of Stx2f in a cell-based assay. These mAbs were also demonstrated to be highly specific and reactive when applied to colony immunoblot assays. Conclusions: Novel mAbs specific to Stx2f were developed for the first time, providing new assets for the STEC community. Immunoassays with improved sensitivity and specificity are demonstrated for the detection of Stx2f present in food, environmental, and clinical samples.