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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Foodborne Toxin Detection and Prevention Research » Research » Publications at this Location » Publication #314483
Title: New Stx2e monoclonal antibodies for immunological detection and distinction of Stx2 subtypes

Author
item Skinner, Craig
item Patfield, Stephanie
item Hernlem, Bradley - Brad
item He, Xiaohua

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/12/2015
Publication Date: 7/20/2015
Publication URL: http://handle.nal.usda.gov/10113/61413
Citation: Skinner, C.B., Patfield, S.A., Hernlem, B.J., He, X. 2015. New Stx2e monoclonal antibodies for immunological detection and distinction of Stx2 subtypes. PLoS One. 10(7):e0132419.

Interpretive Summary: Shiga toxins (Stx) are among the most clinically important virulence factors for enterohemhorragic Escherichia coli (EHEC), and they are diverse with respect to their sequence and pathogenicity. Stx2e is among the eleven subtypes of Shiga toxins, and while Stx2a, 2c, and 2d cause the most severe symptoms in humans, strains of E. coli which express Stx2e are responsible for the potentially fatal edema disease in newborn piglets. Stx2e is poorly recognized by many antibodies that detect Stx2, however, meaning that many Stx2 detection kits cannot detect Stx2e. Four new Stx2e-specific antibodies were therefore generated and characterized, two of which recognize the A subunit of Sx2e, and two which recognize the B subunit. Using these antibodies, we developed an assay which can detect Stx2e at concentrations as low as 11.8 pg/mL, which is the lowest limit of detection reported for Stx2e in an immunoassay to date. The two B-subunit-binding anti-Stx2e antibodies also neutralized toxin in cell toxicity assays. Many strains of EHEC express some combination of Stx2a, Stx2c, and Stx2d and expression of multiple Stx in the same bacteria could cause enhanced pathogenicity. However, there are no commercial immunoassays that can discern the Stx2a, Stx2c, and Stx2d subtypes independently from each other. Using a combination of these and previously generated antibodies, we have developed assays that can detect each of these subtypes individually, contributing important tools to the study of multiple-Stx EHEC.

Technical Abstract: Background Stx2e is a primary virulence factor in STEC strains that cause edema disease in neonatal piglets. Though Stx2a and Stx2e are similar, most antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype. Methods and Findings Four monoclonal antibodies recognizing Stx2e were developed and characterized. Two of these mAbs recognize the B subunit of Stx2e, Stx2f, and to a lesser extent, Stx2b, Stx2c, and Stx2d. The other two mAbs recognize the A subunit of Stx2e, and cross-react with all Stx2 subtypes except Stx2f. The most sensitive sandwich ELISA using these mAbs has a limit of detection for Stx2e of 11.8 pg/mL. The ability of the neutralizing antibody Stx2e-2 to block Stx2e-receptor binding in Vero cells was visualized using immunofluorescence. Combinations of these and previously developed mAbs permit immunological differentiation between closely related Stx2a, Stx2c, and Stx2d. Conclusions The sensitive immunoassays developed in this study should augment our capacity to detect Stx2e in porcine environments and biological samples. Moreover, immunoassays that can distinguish between the closely related Stx2a, Stx2c, and Stx2d subtypes can be useful in quickly analyzing Stx subtypes in samples containing more than one strain of STEC.