Antibodies anti-Shiga toxin 2 B subunit from chicken egg yolk: Isolation, purification and neutralization efficacy

Abstract

Shiga toxins (Stx1 and Stx2) are the main virulence factors of enterohemorrhagic Escherichia coli (EHEC), a foodborne pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The aim of this study was to evaluate the antibodies against Stx2 obtained from egg yolks of laying hens immunized with a recombinant Stx2B subunit. A high specific response in serum was observed 25 days after the first immunization and IgY antibodies were extracted from day 47th and purified from egg yolk. A concentration of 0.84 mg of total IgY/ml of egg yolk was obtained, of which 8% were antigen specific. The ability of anti-Stx2B IgY to recognize Stx2B and Stx2 either in solid-phase or in solution were evaluated and compared with anti-Stx2B rabbit antibodies by Western blotting and ELISA. The protective efficacy of IgY against Stx2 was determined by in vitro and in vivo experiments. The results show that IgY was able to recognize Stx2B and Stx2 in denatured conditions, attached to a solid-phase and free in solution. The anti-Stx2B IgY could effectively block the biological activity of Stx2 on Vero cells and protect mice from Stx2 challenge. The data suggest that immunization of hens with Stx2B could be a strategy to obtain at low cost a relatively high concentration of anti-Stx2 egg yolk IgY, able to neutralize Stx2 lethal activity. IgY technology could be an useful tool for research, diagnosis and therapy of EHEC infection.

Environmental Cues Modulate Microglial Cell Behavior Upon Shiga Toxin 2 From Enterohemorrhagic Escherichia coli Exposure

“…As part of the Stx family, Stx2 has an AB5 structure in which a single subunit A is associated with five subunits B. Subunit A represents the catalytic activity while the B-pentamer (Stx2B) is the binding site of the Stx2-Gb3 receptor (Fraser et al, 2004). Recombinant Stx2B (Parma et al, 2011) was used to compare its biological effects on MG cells with the Stx2 one. Iba1 expression levels equally increased in both Stx2-and Stx2B-incubated MG cells, with or W/O context.…”

Mouse in Vivo Neutralization of Escherichia coli Shiga Toxin 2 with Monoclonal Antibodies

“…Therefore, passive administration of toxin-neutralizing antibodies should be an effective therapy for HUS. A number of Stx-specific mAbs have been developed and tested for their ability to protect animals from Stx-mediated death [23,24,25,26,27,28,29,30,31,32]. However, a detailed toxicokinetic analysis of un-modified Stx2 in the presence or absence of neutralizing antibodies against this toxin in an animal model has not been fully described in the literature.…”

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.…”

“…The two control chickens did not produce any anti-Stx2e antibodies during the experimental period. The antigenicity of the B subunit of Stx (Stx2B) reportedly differs according to the species immunized; rabbits produce Stx2B-specific antibodies, but mice do not (18 (20,21). 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.…”