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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

Receptor binding and neutralization of Stx2e.A) FACS analysis of Gb3-LPS, Gb4-LPS, and control E. coli cells. Stx2e (E167Q) and mAb Stx2e-3 were used at 1 μg/mL, and 50,000 cells were analyzed per sample. B) Administration of mAbs Stx2e-1 or Stx2e-2 at 10 μg/mL protects Vero cells from Stx2e toxicity (Stx2e-containing media at a 10-fold dilution). mAbs Stx2e-3 and Stx2e-4 (also at 10 μg/mL) do not. None of the four mAbs protect Vero cells from Stx2a (Stx2a-containing media at a 10-fold dilution).
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pone.0132419.g004: Receptor binding and neutralization of Stx2e.A) FACS analysis of Gb3-LPS, Gb4-LPS, and control E. coli cells. Stx2e (E167Q) and mAb Stx2e-3 were used at 1 μg/mL, and 50,000 cells were analyzed per sample. B) Administration of mAbs Stx2e-1 or Stx2e-2 at 10 μg/mL protects Vero cells from Stx2e toxicity (Stx2e-containing media at a 10-fold dilution). mAbs Stx2e-3 and Stx2e-4 (also at 10 μg/mL) do not. None of the four mAbs protect Vero cells from Stx2a (Stx2a-containing media at a 10-fold dilution).

Mentions: The primary cellular receptor for Stx2e is thought to be Gb4-Cer [26]; it has been previously shown, however, that Stx2e can bind a wide range of receptors (Gb3, Gb4, Forsman, etc.) [12]. Using Gb3- and Gb4-LPS-expressing E. coli and our new mAbs, we sought to confirm receptor specificities for Stx2e. In FACS analysis, Stx2e (E167Q) bound to both Gb3- and Gb4-LPS-expressing cells, but Stx2a (E167Q) only bound to Gb3-LPS-expressing cells (Fig 4A). Antibodies recognizing the B subunits of Stx frequently neutralize the toxin that they are specific to in cell toxicity assays [13, 16]. Our mAbs against Stx2e were not an exception: both B subunit-specific mAbs (Stx2e-1 and Stx2e-2) protected Vero cells from Stx2e toxicity derived from cell-free culture media, whereas both A subunit-specific mAbs (Stx2e-3 and Stx2e-4) did not (Fig 4B).


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

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

Receptor binding and neutralization of Stx2e.A) FACS analysis of Gb3-LPS, Gb4-LPS, and control E. coli cells. Stx2e (E167Q) and mAb Stx2e-3 were used at 1 μg/mL, and 50,000 cells were analyzed per sample. B) Administration of mAbs Stx2e-1 or Stx2e-2 at 10 μg/mL protects Vero cells from Stx2e toxicity (Stx2e-containing media at a 10-fold dilution). mAbs Stx2e-3 and Stx2e-4 (also at 10 μg/mL) do not. None of the four mAbs protect Vero cells from Stx2a (Stx2a-containing media at a 10-fold dilution).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132419.g004: Receptor binding and neutralization of Stx2e.A) FACS analysis of Gb3-LPS, Gb4-LPS, and control E. coli cells. Stx2e (E167Q) and mAb Stx2e-3 were used at 1 μg/mL, and 50,000 cells were analyzed per sample. B) Administration of mAbs Stx2e-1 or Stx2e-2 at 10 μg/mL protects Vero cells from Stx2e toxicity (Stx2e-containing media at a 10-fold dilution). mAbs Stx2e-3 and Stx2e-4 (also at 10 μg/mL) do not. None of the four mAbs protect Vero cells from Stx2a (Stx2a-containing media at a 10-fold dilution).
Mentions: The primary cellular receptor for Stx2e is thought to be Gb4-Cer [26]; it has been previously shown, however, that Stx2e can bind a wide range of receptors (Gb3, Gb4, Forsman, etc.) [12]. Using Gb3- and Gb4-LPS-expressing E. coli and our new mAbs, we sought to confirm receptor specificities for Stx2e. In FACS analysis, Stx2e (E167Q) bound to both Gb3- and Gb4-LPS-expressing cells, but Stx2a (E167Q) only bound to Gb3-LPS-expressing cells (Fig 4A). Antibodies recognizing the B subunits of Stx frequently neutralize the toxin that they are specific to in cell toxicity assays [13, 16]. Our mAbs against Stx2e were not an exception: both B subunit-specific mAbs (Stx2e-1 and Stx2e-2) protected Vero cells from Stx2e toxicity derived from cell-free culture media, whereas both A subunit-specific mAbs (Stx2e-3 and Stx2e-4) did not (Fig 4B).

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