Dataset Information

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

ABSTRACT: 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.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).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.

SUBMITTER: Skinner C

PROVIDER: S-EPMC4507848 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Skinner Craig C Patfield Stephanie S Hernlem Bradley J BJ He Xiaohua X

PloS one 20150720 7

<h4>Background</h4>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.<h4>Methods and findings</h4>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 ...[more]

PMID: 26192407

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Project description:BackgroundHemolytic uremic syndrome (HUS) leading to acute kidney failure, is a condition linked to the production of primarily Shiga toxin 2 (Stx2) by some E. coli serotypes. We have previously shown that Stx2 A subunit-specific human monoclonal antibody (HuMAb) 5C12, and B subunit-specific HuMAb 5H8 inhibit cultured cell death, and protect mice and piglets from fatal Stx2-intoxication. We have also shown that 5H8 blocks binding of Stx2 to its cell-surface receptor globotriaosyl ceramide (Gb(3)), whereas Stx2 when complexed with 5C12 binds Gb(3) with higher affinity than Stx2. The mechanism by which 5C12 neutralizes Stx2 in vitro involves trapping of Stx2 in the recycling endosomes and releasing it into the extracellular environment. Because of the clinical implications associated with the formation of Stx2/antibody complexes and the potential for trapping and clearance through a severely damaged kidney associated with HUS, we investigated the likely site(s) of Stx2/antibody localization and clearance in intoxicated mice treated with antibody or placebo.ResultsMice were injected with radiolabeled Stx2 ((125)I-Stx2) 4 hours after administration of 5C12, 5H8, or phosphate buffered saline (PBS) and the sites of localization of labeled Stx2, were investigated 3, 24 and 48 hours later. The liver recorded statistically much higher concentrations of labeled Stx2 for groups receiving 5C12 and 5H8 antibodies after 3, 24 and 48 hours, as compared with the PBS group. In contrast, highest levels of labeled Stx2 were detected in the kidneys of the PBS group at all 3 sampling times. Mice receiving either of the two HuMAbs were fully protected against the lethal effect of Stx2, as compared with the fatal outcome of the control group.ConclusionsThe results suggest that HuMAbs 5C12 and 5H8 promoted hepatic accumulation and presumably clearance of toxin/antibody complexes, significantly diverting Stx2 localization in the kidneys, the target of Stx2 and the cause of HUS. This is in contrast to the fatal outcome of the control group receiving PBS. The results also confirm earlier observations that both HuMAbs are highly and equally protective against Stx2 intoxication in mice.

Dataset Information

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

Publications

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

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