Bacterial adhesion to surface-coated vWF

The ability of S. aureus or L. lactis ectopically expressing IsdB to adhere to surface-coated vWF was evaluated by ELISA-based assay. Microtiter wells coated with vWF were incubated with cells (A₆₀₀ = 1.0) of S. aureus SH1000 and its isdB mutant obtained from cultures grown to stationary phase in RPMI and suspended in 0.5% (v/v) BSA. The wells were extensively washed with PBST, blocked with 2% (v/v) BSA, and incubated with 100 µl bacterial suspensions for 1 h at 22 °C. The expression of SpA on cell surface was exploited to detect bacteria adhesion by incubating the plates for 45 min with HRP-conjugated rabbit anti-mouse IgG (1:1000).

Binding of HRP-conjugated rabbit anti-mouse IgG (1:1000) to surface-coated S. aureus SH1000 and its isdB isogenic mutant cells (1 × 10⁷) was measured in a standard ELISA assay.

Binding of non-immune IgG such as HRP-conjugated goat anti-rabbit IgG or anti-L. lactis IgG to IsdB was determined by incubating surface-coated recombinant IsdB (1 μγ/well) with the antibodies and antibody binding was detected as reported above.

Adhesion of L. lactis to vWF was performed by incubating plates coated with vWF with cells of L. lactis expressing IsdB (L. lactis _{pNZ8037::isdB}) and the strain carrying the empty vector (L.lactis _pNZ8037) obtained from cultures grown in BHI. L. lactis binding to surface-coated vWF was detected by incubating for 1 h at 22 °C with rabbit polyclonal anti-L. lactis IgG (1 µg/well) followed by an HRP-conjugated goat anti-rabbit IgG (1:1000).