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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

Stx2a-, Stx2c-, and Stx2d-specific ELISAs.A) A Stx2a-specific ELISA (Stx2-5 used for capture; Stx2-1 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d. B) A Stx2c-specific ELISA (Stx2-5 for capture; Stx2e-2 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d. C) A Stx2d-specific ELISA (Stx2e-3 for capture; Stx2e-2 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d.
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pone.0132419.g003: Stx2a-, Stx2c-, and Stx2d-specific ELISAs.A) A Stx2a-specific ELISA (Stx2-5 used for capture; Stx2-1 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d. B) A Stx2c-specific ELISA (Stx2-5 for capture; Stx2e-2 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d. C) A Stx2d-specific ELISA (Stx2e-3 for capture; Stx2e-2 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d.

Mentions: In an effort to find antibody combinations that could distinguish Stx2 subtypes, four antibodies described in the study were used with two previously described mAbs, Stx2-1 and Stx2-5, derived from Stx2a [13] in ELISAs. All possible antibody combinations using Stx2e-1, Stx2e-2, Stx2e-3, Stx2e-4, Stx2-1, and Stx2-5 for capture and/or detection were evaluated using cell-free media from all seven Stx2 subtypes (pure toxins are currently not available for some subtypes of Stx2). Among the combinations of antibodies, mAbs Stx2-5/Stx2-1 (capture/detection) appeared to recognize only Stx2a (S1 Table). The Stx2-5/Stx2e-2 combination had good affinity for Stx2c and recognized Stx2d weakly, but did not detect Stx2a. The combination of Stx2e-3/Stx2e-2 was very effective at detecting Stx2d and Stx2e, detected Stx2c poorly, and did not recognize Stx2a. These three antibody combinations were then evaluated for their efficacy at distinguishing Stx2a, Stx2c, and Stx2d. Using pure Stx2a, Stx2c, and Stx2d, standard curves were conducted with all three ELISA combinations (Fig 3). As expected, the Stx2-5/Stx2-1 antibody combination was specific to Stx2a only (Fig 3A) (with a LOD of 0.13 ng.mL). The Stx2-5/Stx2e-2 combination recognized Stx2c well (LOD = 0.78 ng/mL), Stx2d poorly (LOD = 0.9 ng/mL), and did not detect Stx2a (Fig 3B). The Stx2e-3/Stx2e-2 combination was very effective at detecting Stx2d (LOD = 0.23 ng/mL), very poor at detecting Stx2c (LOD = 51.2 ng/mL), and did not detect Stx2a at all (Fig 3C).


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

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

Stx2a-, Stx2c-, and Stx2d-specific ELISAs.A) A Stx2a-specific ELISA (Stx2-5 used for capture; Stx2-1 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d. B) A Stx2c-specific ELISA (Stx2-5 for capture; Stx2e-2 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d. C) A Stx2d-specific ELISA (Stx2e-3 for capture; Stx2e-2 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d.
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pone.0132419.g003: Stx2a-, Stx2c-, and Stx2d-specific ELISAs.A) A Stx2a-specific ELISA (Stx2-5 used for capture; Stx2-1 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d. B) A Stx2c-specific ELISA (Stx2-5 for capture; Stx2e-2 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d. C) A Stx2d-specific ELISA (Stx2e-3 for capture; Stx2e-2 for detection) was conducted on pure Stx2a, Stx2c, and Stx2d.
Mentions: In an effort to find antibody combinations that could distinguish Stx2 subtypes, four antibodies described in the study were used with two previously described mAbs, Stx2-1 and Stx2-5, derived from Stx2a [13] in ELISAs. All possible antibody combinations using Stx2e-1, Stx2e-2, Stx2e-3, Stx2e-4, Stx2-1, and Stx2-5 for capture and/or detection were evaluated using cell-free media from all seven Stx2 subtypes (pure toxins are currently not available for some subtypes of Stx2). Among the combinations of antibodies, mAbs Stx2-5/Stx2-1 (capture/detection) appeared to recognize only Stx2a (S1 Table). The Stx2-5/Stx2e-2 combination had good affinity for Stx2c and recognized Stx2d weakly, but did not detect Stx2a. The combination of Stx2e-3/Stx2e-2 was very effective at detecting Stx2d and Stx2e, detected Stx2c poorly, and did not recognize Stx2a. These three antibody combinations were then evaluated for their efficacy at distinguishing Stx2a, Stx2c, and Stx2d. Using pure Stx2a, Stx2c, and Stx2d, standard curves were conducted with all three ELISA combinations (Fig 3). As expected, the Stx2-5/Stx2-1 antibody combination was specific to Stx2a only (Fig 3A) (with a LOD of 0.13 ng.mL). The Stx2-5/Stx2e-2 combination recognized Stx2c well (LOD = 0.78 ng/mL), Stx2d poorly (LOD = 0.9 ng/mL), and did not detect Stx2a (Fig 3B). The Stx2e-3/Stx2e-2 combination was very effective at detecting Stx2d (LOD = 0.23 ng/mL), very poor at detecting Stx2c (LOD = 51.2 ng/mL), and did not detect Stx2a at all (Fig 3C).

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