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 stx2EDL933, stx2vha, stx2vhb, stx2g, stx1EDL933, and stx1d 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.

Sensitive detection of active Shiga toxin using low cost CCD based optical detector

“…However, sandwich ELISA detection limit for Stx2 is 115 ng/ml (Parma, Y.R., et al, 2012) while the level of detection reported here is 0.1 pg/mL of Stx2 so the CCD sensitivity is sufficient compared with current assays. Moreover, photomultipliers are inherently spot detectors, with analysis of limited size areas so in most devices the plate must be scanned by the after heat inactivation) is non toxic, so the novel approach described here is more clinically relevant than the published work.…”

Evaluation of Shiga toxin 2e‐specific chicken egg yolk immunoglobulin: Production and neutralization activity

“…Conversely, Parma et al . prepared Stx2B‐specific IgY by immunizing chickens with recombinant Stx2B and confirmed its specificity by western blotting . For Stx2e, the chickens clearly produced a B subunit‐specific IgY that could detect the B subunit even under western blotting conditions, suggesting that chicken IgY, rather than rabbit antibodies, can be used to detect Stx2e.…”

Recent trends in the detection of pathogenic Escherichia coli O157 : H7

“…A good example is the study 29 by Ferreira et al in which a mouse assay and a modified ELISA test were used to detect nanograms of Clostridium botulinum verotoxin in hash brown potatoes. More recently 30 Parma et al developed a sandwich ELISA for the determination of Shiga toxin (Stx) using anti-Stx2B subunit antibodies. Its performance was compared with that of the Vero cell assay and a commercial immunoassay kit.…”