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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 antibody specificity.A) Direct ELISA using Stx2e mAbs and purified Stx toxoids. Stx2e mAb was added at 1 μg/mL; purified toxoids at 100 ng/mL. B) Western immunoblots for mAbs Stx2e-1, Stx2e-2, and Stx2e-3. Media from Stx-expressing strains were loaded at 10 μL/lane. The “No Stx” sample contains media from an E. coli strain that doesn’t express Stx (ATCC 25922). C) Stx2e B subunit ELISA. Direct ELISA with Stx2e B subunit bound to ELISA plates at 1 μg/mL. Antibodies were also used at 1 μg/mL.
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pone.0132419.g001: Stx2e antibody specificity.A) Direct ELISA using Stx2e mAbs and purified Stx toxoids. Stx2e mAb was added at 1 μg/mL; purified toxoids at 100 ng/mL. B) Western immunoblots for mAbs Stx2e-1, Stx2e-2, and Stx2e-3. Media from Stx-expressing strains were loaded at 10 μL/lane. The “No Stx” sample contains media from an E. coli strain that doesn’t express Stx (ATCC 25922). C) Stx2e B subunit ELISA. Direct ELISA with Stx2e B subunit bound to ELISA plates at 1 μg/mL. Antibodies were also used at 1 μg/mL.

Mentions: Recombinant catalytically-inactive Stx2e toxoid harboring the E167Q point mutation was generated, expressed in E. coli, and purified by column chromatography (a listing of all bacterial strains used and generated in this study is provided in Table 1). This antigen was injected into mice and, using standard hybridoma techniques, splenocytes were extracted and fused to SP2/0 myeloma cells. A total of 1,920 wells of hybridomas were screened for antibodies that recognize Stx2e. After three rounds of clonal selection and recovery, four hybridomas that produced high-affinity monoclonal antibodies (mAbs) to Stx2e were isolated (Fig 1A). The isotype and dissociation constants for these mAbs are displayed in Table 2. mAbs Stx2e-1 and Stx2e-2 recognize the B subunits of Stx2e and Stx2f in a western immunoblot (Fig 1B), although they both can also detect Stx2b, Stx2c, and Stx2d in same-antibody sandwich ELISAs (Stx2e-1 or Stx2e-2 used for both capture and detection) (S1 Fig). mAb Stx2e-3 detects the A-subunit of all Stx2 subtypes except for Stx2f (Fig 1B). mAb Stx2e-4 did not recognize any Stx subtypes, even Stx2e, when analyzed by western immunoblot (data not shown). However, it does recognize Stx2e (E167Q) in a direct ELISA, as do the other three Stx2e mAbs (Fig 1A). Most likely, mAb Stx2e-4 recognizes a conformational epitope on Stx2e which is disrupted during SDS treatment. mAb Stx2e-4 does not recognize recombinant Stx2e B subunit (Fig 1C) in an ELISA, meaning that this is most likely an A subunit-specific antibody.


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

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

Stx2e antibody specificity.A) Direct ELISA using Stx2e mAbs and purified Stx toxoids. Stx2e mAb was added at 1 μg/mL; purified toxoids at 100 ng/mL. B) Western immunoblots for mAbs Stx2e-1, Stx2e-2, and Stx2e-3. Media from Stx-expressing strains were loaded at 10 μL/lane. The “No Stx” sample contains media from an E. coli strain that doesn’t express Stx (ATCC 25922). C) Stx2e B subunit ELISA. Direct ELISA with Stx2e B subunit bound to ELISA plates at 1 μg/mL. Antibodies were also used at 1 μg/mL.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132419.g001: Stx2e antibody specificity.A) Direct ELISA using Stx2e mAbs and purified Stx toxoids. Stx2e mAb was added at 1 μg/mL; purified toxoids at 100 ng/mL. B) Western immunoblots for mAbs Stx2e-1, Stx2e-2, and Stx2e-3. Media from Stx-expressing strains were loaded at 10 μL/lane. The “No Stx” sample contains media from an E. coli strain that doesn’t express Stx (ATCC 25922). C) Stx2e B subunit ELISA. Direct ELISA with Stx2e B subunit bound to ELISA plates at 1 μg/mL. Antibodies were also used at 1 μg/mL.
Mentions: Recombinant catalytically-inactive Stx2e toxoid harboring the E167Q point mutation was generated, expressed in E. coli, and purified by column chromatography (a listing of all bacterial strains used and generated in this study is provided in Table 1). This antigen was injected into mice and, using standard hybridoma techniques, splenocytes were extracted and fused to SP2/0 myeloma cells. A total of 1,920 wells of hybridomas were screened for antibodies that recognize Stx2e. After three rounds of clonal selection and recovery, four hybridomas that produced high-affinity monoclonal antibodies (mAbs) to Stx2e were isolated (Fig 1A). The isotype and dissociation constants for these mAbs are displayed in Table 2. mAbs Stx2e-1 and Stx2e-2 recognize the B subunits of Stx2e and Stx2f in a western immunoblot (Fig 1B), although they both can also detect Stx2b, Stx2c, and Stx2d in same-antibody sandwich ELISAs (Stx2e-1 or Stx2e-2 used for both capture and detection) (S1 Fig). mAb Stx2e-3 detects the A-subunit of all Stx2 subtypes except for Stx2f (Fig 1B). mAb Stx2e-4 did not recognize any Stx subtypes, even Stx2e, when analyzed by western immunoblot (data not shown). However, it does recognize Stx2e (E167Q) in a direct ELISA, as do the other three Stx2e mAbs (Fig 1A). Most likely, mAb Stx2e-4 recognizes a conformational epitope on Stx2e which is disrupted during SDS treatment. mAb Stx2e-4 does not recognize recombinant Stx2e B subunit (Fig 1C) in an ELISA, meaning that this is most likely an A subunit-specific antibody.

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