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New Stx2e Monoclonal Antibodies for Immunological Detection and Distinction of Stx2 Subtypes.

Skinner C, Patfield S, Hernlem BJ, He X - PLoS ONE (2015)

Bottom Line: Though Stx2a and Stx2e are similar, many antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype.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.

View Article: PubMed Central - PubMed

Affiliation: Western Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, California, United States of America.

ABSTRACT

Background: Stx2e is a primary virulence factor in STEC strains that cause edema disease in neonatal piglets. Though Stx2a and Stx2e are similar, many antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype.

Methods and findings: Four monoclonal antibodies against 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 ELISA-based differentiation between closely related Stx2a, Stx2c, and Stx2d (using mAbs Stx2-5/2-1, Stx2-5/2e-2, and Stx2e-3/2e-2, respectively).

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.

No MeSH data available.


Related in: MedlinePlus

Immunofluorescence of Stx2e (E167Q) in Vero cells.mAb Stx2e-3 (1 μg/mL) was used to visualize Stx2e (E167Q) (1 μg/mL) in Vero cells. Stx2e (E167Q) was pre-incubated with either neutralizing mAb Stx2e-2 in media or media alone before adding to Vero cells. Cells were either fixed in paraformaldehyde before Stx2e (E167Q) treatment or after a 0 or 3 hour incubation with Stx2e (E167Q). After fixing and Stx2e (E167Q) treatment, the cells were blocked with BSA, incubated with mAb Stx2e-3, then incubated with anti-mouse DyLight (green images) and DAPI (to stain nuclei, blue images).
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pone.0132419.g005: Immunofluorescence of Stx2e (E167Q) in Vero cells.mAb Stx2e-3 (1 μg/mL) was used to visualize Stx2e (E167Q) (1 μg/mL) in Vero cells. Stx2e (E167Q) was pre-incubated with either neutralizing mAb Stx2e-2 in media or media alone before adding to Vero cells. Cells were either fixed in paraformaldehyde before Stx2e (E167Q) treatment or after a 0 or 3 hour incubation with Stx2e (E167Q). After fixing and Stx2e (E167Q) treatment, the cells were blocked with BSA, incubated with mAb Stx2e-3, then incubated with anti-mouse DyLight (green images) and DAPI (to stain nuclei, blue images).

Mentions: Immunofluoresence is a useful method to track Stx as it progresses from cell surface binding to its target cellular compartments. Interactions of Stx1 and Stx2 with Vero cells have been demonstrated successfully using FITC-labeled anti-mouse IgG secondary antibody [27]. In this study, we demonstrate that the Stx2e (E167Q) toxoid is capable of binding receptors on the surface of Vero cells by first fixing the cells, then treating the cells with the toxoid using immunofluorescence assays. Cells without toxoid treatment were used as a negative control and no green fluorescent signals were observed for these cells (Fig 5, “Stx2e added after fixing” column). To determine if Stx2e (E167Q) toxoid is capable of entering the cells, Vero cells were treated with Stx2e (E167Q) for three hours prior to fixing. As shown in Fig 5, Stx2e (E167Q) entered the cells successfully, and appears to spread throughout the cytoplasm but concentrate around the nucleus. It has been suggested that anti-Stx2e single-chain antibodies can neutralize Stx2e toxicity by blocking the binding of Stx2e to cellular receptors [16]. In agreement with this report, we found that pre-incubation of Stx2e (E167Q) with our neutralizing mAb Stx2e-2 disabled the toxoid binding to Vero cells as well (Fig 5, “Stx2e pre-incubated with Stx2e-2” column). Our immunofluorescence assays also indicate that the mAb Stx2e-3 is able to recognize the Stx2e (E167Q) interacting with cells, regardless of whether the toxoid is internal or external.


New Stx2e Monoclonal Antibodies for Immunological Detection and Distinction of Stx2 Subtypes.

Skinner C, Patfield S, Hernlem BJ, He X - PLoS ONE (2015)

Immunofluorescence of Stx2e (E167Q) in Vero cells.mAb Stx2e-3 (1 μg/mL) was used to visualize Stx2e (E167Q) (1 μg/mL) in Vero cells. Stx2e (E167Q) was pre-incubated with either neutralizing mAb Stx2e-2 in media or media alone before adding to Vero cells. Cells were either fixed in paraformaldehyde before Stx2e (E167Q) treatment or after a 0 or 3 hour incubation with Stx2e (E167Q). After fixing and Stx2e (E167Q) treatment, the cells were blocked with BSA, incubated with mAb Stx2e-3, then incubated with anti-mouse DyLight (green images) and DAPI (to stain nuclei, blue images).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507848&req=5

pone.0132419.g005: Immunofluorescence of Stx2e (E167Q) in Vero cells.mAb Stx2e-3 (1 μg/mL) was used to visualize Stx2e (E167Q) (1 μg/mL) in Vero cells. Stx2e (E167Q) was pre-incubated with either neutralizing mAb Stx2e-2 in media or media alone before adding to Vero cells. Cells were either fixed in paraformaldehyde before Stx2e (E167Q) treatment or after a 0 or 3 hour incubation with Stx2e (E167Q). After fixing and Stx2e (E167Q) treatment, the cells were blocked with BSA, incubated with mAb Stx2e-3, then incubated with anti-mouse DyLight (green images) and DAPI (to stain nuclei, blue images).
Mentions: Immunofluoresence is a useful method to track Stx as it progresses from cell surface binding to its target cellular compartments. Interactions of Stx1 and Stx2 with Vero cells have been demonstrated successfully using FITC-labeled anti-mouse IgG secondary antibody [27]. In this study, we demonstrate that the Stx2e (E167Q) toxoid is capable of binding receptors on the surface of Vero cells by first fixing the cells, then treating the cells with the toxoid using immunofluorescence assays. Cells without toxoid treatment were used as a negative control and no green fluorescent signals were observed for these cells (Fig 5, “Stx2e added after fixing” column). To determine if Stx2e (E167Q) toxoid is capable of entering the cells, Vero cells were treated with Stx2e (E167Q) for three hours prior to fixing. As shown in Fig 5, Stx2e (E167Q) entered the cells successfully, and appears to spread throughout the cytoplasm but concentrate around the nucleus. It has been suggested that anti-Stx2e single-chain antibodies can neutralize Stx2e toxicity by blocking the binding of Stx2e to cellular receptors [16]. In agreement with this report, we found that pre-incubation of Stx2e (E167Q) with our neutralizing mAb Stx2e-2 disabled the toxoid binding to Vero cells as well (Fig 5, “Stx2e pre-incubated with Stx2e-2” column). Our immunofluorescence assays also indicate that the mAb Stx2e-3 is able to recognize the Stx2e (E167Q) interacting with cells, regardless of whether the toxoid is internal or external.

Bottom Line: Though Stx2a and Stx2e are similar, many antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype.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.

View Article: PubMed Central - PubMed

Affiliation: Western Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, California, United States of America.

ABSTRACT

Background: Stx2e is a primary virulence factor in STEC strains that cause edema disease in neonatal piglets. Though Stx2a and Stx2e are similar, many antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype.

Methods and findings: Four monoclonal antibodies against 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 ELISA-based differentiation between closely related Stx2a, Stx2c, and Stx2d (using mAbs Stx2-5/2-1, Stx2-5/2e-2, and Stx2e-3/2e-2, respectively).

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.

No MeSH data available.


Related in: MedlinePlus