Table 1.
Detection of Stx2 in culture supernatants of STEC strains.
Table 2.
Results of PCR and ELISA for detection of Stx2 in E. coli strains isolated from human and environmental samples.
Figure 1.
Activity of antiserum from pre-immune (PI) rabbits, 1st bleeding (one week after 3rd immunization), 2nd bleeding (two weeks after 3rd immunization), 3rd bleeding (one week after boost with additional immunogen), and 4th bleeding (three weeks after boost).
ELISA was performed using Sifin 2B as a capture antibody (1 μg/mL), Stx2 toxoid (10 ng/mL), and antisera of rabbits 5153 (A) and 5154 (B) diluted in a range of 1∶1,000–1∶8,000. Each column represents the mean of 3 independent repeats performed in duplicates, the standard deviation for each column ranges from 0.01 to 0.15.
Figure 2.
Reactivity of immunized rabbit serum IgG to Stx2a.
Lane 1, Coomassie stained SDS-PAGE with 1 μg of purified Stx2a; lanes 2 and 3, Western blot of 0.5 μg of purified Stx2a probed with a mixture of mouse mAbs against Stx2 A- and B-subunits and rabbit 5154 serum IgG followed by horseradish peroxidase-conjugated goat secondary antibodies against mouse or rabbit IgG. The A and B subunit positions are indicated by arrows.
Table 3.
Toxin specificity of the polyclonal antibody tested by direct ELISA.
Figure 3.
Neutralization of Stx2a cytotoxicity with the pAb.
Vero cells were incubated in DMEM medium containing Stx2a (10 ng/mL) with or without the presence of the pAb. The cytotoxicity to cells was calculated as [(cps from negative control – cps from samples treated)/cps from negative control] ×100. The luminescent counts from cells grown in DMEM medium were used as a negative control. The relative cytotoxicity was calculated by normalizing each value to the cytotoxicity of Stx2a without adding pAb as 100%. The results represent the mean ± SD of three replicates from one representative experiment. Three individual experiments were performed.
Figure 4.
Comparison of antigen binding capacity between pAb derived from RB 5154 and commercially available mAbs, Sifin2A and Sifin2B.
Coating antigen (Stx2a) was diluted in PBS at concentrations indicated. Primary antibodies (pAb or mAb) were used at 1 µg/mL and secondary antibodies (goat anti-rabbit or goat anti-mouse IgG) conjugated with HRP were used at 1∶5,000. Each bar represents the mean of three independent repeats performed in duplicates.
Figure 5.
Detection of Stx2a spiked in water (♦) and broth of soil (•) and feces (▴) by a sandwich ELISA using Stx2-pAb as capture antibody (1 µg/mL) and HRP conjugated Stx2-pAb as detector antibody (100 ng/mL).
Data represent the average of three determinations ± SD.
Table 4.
Stx2 polyclonal antisera binding to consensus epitopes of A-subunit determined using overlapping Stx2a peptides.
Table 5.
Stx2 polyclonal antisera binding to consensus epitopes of B-subunit determined using overlapping Stx2a peptides.