3.1 | Binding affinity and specificity of mAb-EspB-B7 to EspB
To evaluate the binding affinity of mAb-EspB-B7 to EspB, we performed ELISA (Figure 1A) and SPR (Figure 1B) binding assays. We found that mAb-EspB-B7 binds EspB with high affinity, with a KDvalue of 17.4 nM. To determine whether mAb-EspB-B7 binds specifically to EspB under native conditions, we examined the binding of mAb-EspB-B7 to WT EPEC, ΔescN (a mutant lacking a functional T3SS), ΔespB(a mutant that does not express and secrete EspB), and ΔespB +EspB-His (an espB null strain that overexpresses plasmid-encoded EspB-His). The different strains grown under T3SS-inducing conditions were separated into bacterial pellets and supernatants, which were then analyzed by SDS-PAGE and western blot analysis using mAb-EspB-B7 in the detection phase. In agreement with a previous study, we detected significant secretion of EspB into the bacterial supernatant of WT EPEC, but not into the supernatants of the ΔescN or ΔespB mutants (Figure 2A) (Luo & Donnenberg, 2006). Moreover, we observed that over-expression of EspB (ΔespB+ EspB-His) resulted in a higher expression of the protein within the bacteria (pellet) as well as a higher secretion into the extracellular medium (Figure 2A).
3.2 | mAb-EspB-B7 binding to EspB in the assembled