Development of monoclonal antibodies and immunoassays for sensitive and specific detection of Shiga toxin Stx2f

Abstract

Background: Shiga toxin 2 (Stx2) is a major virulence factor in gastrointestinal diseases caused by Escherichia coli. Although Stx2a (prototypical Stx2) is well-studied, all seven subtypes of Stx2 have been associated with disease in mammals. Several subtypes of Stx2, including Stx2f, are difficult to detect immunologically.

Methods and findings: Four novel monoclonal antibodies (mAbs) against the Stx2f subtype were produced and characterized. These mAbs react exclusively to the Stx2f A subunit, and do not cross-react with other subtypes of Stx2. A Stx2f-specific sandwich ELISA was established and a limit of detection of 0.123 ng/mL was obtained using one pair of the mAbs. The receptor preference of Stx2f was confirmed using this sandwich ELISA. Three out of four mAbs can partially neutralize the toxicity of Stx2f in a cell-based assay. These mAbs were also demonstrated to be highly specific and reactive when applied to colony immunoblot assays.

Conclusions: Novel mAbs specific to Stx2f were developed for the first time, providing new assets for the STEC community. Immunoassays with improved sensitivity and specificity will be useful for the detection of Stx2f present in food, environmental, and clinical samples.

Figure 1. Detection of Stx2f by western blot analysis.

A. Undiluted mitomycin C-induced (50 ng/mL) bacterial supernatants containing Stx2f, Stx2a, and K12 were loaded at 5 μL/lane. Purified Stx2f and Stx2a proteins were loaded at 5 ng/lane. Proteins were separated by SDS-PAGE. Membranes were probed with mAbs Stx2f-1, Stx2f-2, Stx2f-3, and Stx2f-4, respectively. Representative blots are shown (N=5). B. Direct ELISA for detection of Stx2f using Stx2f mAbs indicated. A concentration of 250 ng/mL purified Stx2f, Stx2a, and partially purified Stx1 toxin were used to coat the ELISA wells. The concentrations of mAbs and goat anti-mouse-HRP used were 1 µg/mL and 0.2 µg/mL, respectively. Sifin 2A (a mAb specific to Stx2) and Sifin 1 (a mAb specific to Stx1) were included as positive controls. The data shown represent the mean ± SD of three replicates from one representative experiment. Three individual experiments were performed. C. Stx2f sandwich ELISAs comparing different Stx2f antibody pairs. Coating antibodies and biotinylated detection antibodies were used at 1 µg/mL, streptavidin-HRP conjugate was used at 0.1 µg/mL, and the antigen (purified Stx2f) was used at 10 ng/mL. The data shown represent the mean ± SD of three replicates from one representative experiment. Three individual experiments were performed.

Figure 2. Sensitivity and specificity of Stx2f mAbs.

A. Detection of Stx2f in PBS (●) or chicken extract (■) by ELISA using the mAb Stx2f-1 as capture and mAb Stx2f-4 as a detector. Purified Stx2f ranging from 0-60 µg/mL were used for this assay. The data shown represent the mean ± SD of three replicates from one representative experiment (this experiment was performed four times with similar results). B. The mAb Stx2f-1/4 sandwich ELISA reacts exclusively with Stx2f cell culture supernatant. Mitomycin C-induced cell-free bacterial supernatants (at a 2-fold dilution) for all seven subypes of Stx2 were prepared and analyzed by ELISA. The data shown represent the mean ± SD of three replicates from one representative experiment. Three individual experiments were performed.

Figure 3. Binding of Stx2f to Gb3-LPS and Gb4-LPS receptors.

Various amounts of Gb3-LPS- or Gb4-LPS-expressing cells were mixed with a fixed amount of purified Stx2f or Stx2a (250 pg/mL) in a microtube. Unbound toxins were recovered after centrifugation and quantified by ELISA using a mAb pair (Stx2f-1/4 for Stx2f or Sifin A/B for Stx2a). Stx2f binds to Gb3-LPS and Gb4-LPS cells with equal affinity, while Stx2a binds only Gb3-LPS cells. The average of three replicates of a representative experiment is shown (this experiment was conducted three times).

Figure 4. Neutralization of Stx2f by anti-Stx2f mAbs.

Stx2f (5 ng/mL) was pre-incubated with antibody (100 µg/mL) for 1 hour at RT. This mixture was then incubated with Vero cells for 1 hour at 4°C. The media was removed and new media was added. Cell viability was measured using the CellTitre-Glo reagent. Data shown represent the mean ± SD of three replicates from one representative experiment. This experiment was conducted three times with similar results.

Figure 5. Stx2f colony immunoblot and matrix effects.

A. A Stx2f colony immunoblot using mAb Stx2f-4 was conducted upon a mixture of Stx2f-expressing cells and GFP-labeled control cells. The cells were diluted 10⁶ and plated on LB +50 ng/mL mitomycin C. The same petri dish portion is displayed for all four panels (Petri dish, GFP, Stx2f blot, and Overlay). B. A Stx2f colony immunoblot is shown using the same mixture of cells as in 5A, plated on an LB plate containing mitomycin C and supplemented with 50 µL of chicken breast extract. The same petri dish portion is displayed for these four panels.