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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

Stx2e ELISA sensitivity and specificity.A) Optimizing sandwich ELISA antibody combinations. Antibodies were used at 1 μg/mL for capture and 0.5 μg/mL for detection; Stx2e (E167Q) was used at 10 ng/mL in PBS. B) Standard curve for the most sensitive anti-Stx2e sandwich ELISA. Stx2e-3 was used for capture (1 μg/mL); Stx2e-2 was used for detection (0.5 μg/mL). Purified Stx2e (E167Q) toxoid was used as the antigen. The standard curves were linear from 8 to 0.01 ng/mL.
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pone.0132419.g002: Stx2e ELISA sensitivity and specificity.A) Optimizing sandwich ELISA antibody combinations. Antibodies were used at 1 μg/mL for capture and 0.5 μg/mL for detection; Stx2e (E167Q) was used at 10 ng/mL in PBS. B) Standard curve for the most sensitive anti-Stx2e sandwich ELISA. Stx2e-3 was used for capture (1 μg/mL); Stx2e-2 was used for detection (0.5 μg/mL). Purified Stx2e (E167Q) toxoid was used as the antigen. The standard curves were linear from 8 to 0.01 ng/mL.

Mentions: In order to determine which combination of Stx2e mAbs provides the greatest sensitivity for Stx2e, we analyzed all possible combinations of the four Stx2e mAbs that we generated. Almost all combinations were effective at detecting Stx2e (Fig 2A). Although the Stx2e-1/2 and Stx2e-2/1 antibody combinations gave the strongest ELISA signal at a Stx2e concentration of 10 ng/mL (p-value < 0.01), the ELISA comprised of the Stx2e-3/Stx2e-2 combination was the most sensitive due to its lower background, with a limit of detection (LOD) of just 11.8 pg/mL (Fig 2B).


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

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

Stx2e ELISA sensitivity and specificity.A) Optimizing sandwich ELISA antibody combinations. Antibodies were used at 1 μg/mL for capture and 0.5 μg/mL for detection; Stx2e (E167Q) was used at 10 ng/mL in PBS. B) Standard curve for the most sensitive anti-Stx2e sandwich ELISA. Stx2e-3 was used for capture (1 μg/mL); Stx2e-2 was used for detection (0.5 μg/mL). Purified Stx2e (E167Q) toxoid was used as the antigen. The standard curves were linear from 8 to 0.01 ng/mL.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4507848&req=5

pone.0132419.g002: Stx2e ELISA sensitivity and specificity.A) Optimizing sandwich ELISA antibody combinations. Antibodies were used at 1 μg/mL for capture and 0.5 μg/mL for detection; Stx2e (E167Q) was used at 10 ng/mL in PBS. B) Standard curve for the most sensitive anti-Stx2e sandwich ELISA. Stx2e-3 was used for capture (1 μg/mL); Stx2e-2 was used for detection (0.5 μg/mL). Purified Stx2e (E167Q) toxoid was used as the antigen. The standard curves were linear from 8 to 0.01 ng/mL.
Mentions: In order to determine which combination of Stx2e mAbs provides the greatest sensitivity for Stx2e, we analyzed all possible combinations of the four Stx2e mAbs that we generated. Almost all combinations were effective at detecting Stx2e (Fig 2A). Although the Stx2e-1/2 and Stx2e-2/1 antibody combinations gave the strongest ELISA signal at a Stx2e concentration of 10 ng/mL (p-value < 0.01), the ELISA comprised of the Stx2e-3/Stx2e-2 combination was the most sensitive due to its lower background, with a limit of detection (LOD) of just 11.8 pg/mL (Fig 2B).

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