Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

Abstract

Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx(2EDL933), stx(2vha), stx(2vhb), stx(2g), stx(1EDL933), and stx(1d) were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli.

Keywords: ELISA; Escherichia coli; IgY; STEC; Shiga toxin; chicken; egg yolk.

Figure 1

Analysis of Stx2 holotoxin by SDS-PAGE and Western blot. (A) SDS-PAGE (12.5% acrylamide, under reducing conditions) Lane 1: Purified recombinant Stx2B (4.34 μg) Lane 2: Stx2 holotoxin in the eluate (13.5 μl) Lane 3: Stx2 supernatant from strain 59-2 (13.5 μl). Gel was stained with Coomassie Blue. (B) Western blot Lane 1: Purified recombinant Stx2B (4.34 μg) Lane 2: Stx2 holotoxin in the eluate (13.5 μl) Lane 3: Stx2 supernatant from strain 59-2 (13.5 μl). Membrane was incubated with anti-Stx2B IgY and anti-IgY HRP. Arrow: Stx2B subunit position.

Figure 2

Titration curve of purified Stx2 by anti-Stx2B IgY sandwich ELISA. The reaction was carried out using anti-Stx2B IgY followed by serial twofold dilutions of purified Stx2 and incubation with horseradish peroxidase-conjugated anti-Stx2B IgG. The detection limit was calculated from the mean + 3 SD of the blank control.

Figure 3

Interactive dot diagram (MedCalc Stadistical Software). In the graph the data of the Stx-positive and Stx-negative strains are displayed as dots on two vertical axes. A horizontal line indicates the cut-off point with the best separation (minimal false negative and false positive results) between the two groups. The corresponding test characteristics sensitivity and specificity are shown at the right side of the graph.