5 tetramethylbenzidine tmb substrate

The dialyzed 80% ammonium sulfate precipitates from broth cultures of isolates 5HP, XN87, and VPS02 mentioned above were also subjected to ELISA for quantification of the PirB^Vp toxin. The purified rPirB^Vp protein was used to construct a standard curve. Each well of a 96-well plate was coated with 200 μl of coating buffer (0.05 M carbonate buffer, pH 9.6) containing serially diluted standard protein and exposed to the antibody overnight at 4°C. The plates were washed three times with washing buffer (1× phosphate-buffered saline [PBS] containing 0.05% [vol/vol] Tween 20, pH 7.4) and then blocked for 15 min at room temperature with 300 μl of blocking buffer (3% [wt/vol] bovine serum albumin in 1× PBS, pH 7.4). After the washing steps, the plates were incubated with the anti-PirB^Vp MAb followed by goat anti-mouse immunoglobulin with an HRP-conjugated secondary. Bound antibodies were detected by adding 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma), and the reactions were stopped by adding 2 N H₂SO₄. The optical density at 450 nm (OD₄₅₀) was measured using a microplate reader (SpectraMax).

E. coli BL21 (DE3) (Merck) cells containing the pAraBAD-Z-EC vector were cultivated as described above, and induced with 0.6% L-arabinose at 25°C overnight. A non-induced sample was included as a negative control. Cells were labeled with biotinylated IgG as described for the flow-cytometric analysis, followed by streptavidin-HRP (Dako, Glostrup, Denmark) as a secondary reagent. Detection of IgG binding was performed by resuspending the cells in 3,3′, 5,5′ Tetramethylbenzidine (TMB) substrate (Sigma), and the absorbance was measured at 370 nm using a Fluostar Omega plate reader (BMG Labtech, Germany). The experiment was performed in triplicates.

HUVEC were cultured to confluence in 96-well plates and fixed in paraformaldehyde (2%), 15 minutes, room temperature. Cells were washed 3 times in PBS and blocked in 1% BSA, 1 hour, room temperature. rScpA was added to cells in PBS or PBS-FCS (10%), in doubling dilutions from 250 ng. Bound protein was detected following incubation with mouse anti-ScpA serum (1:1000) for 2 hours at room temperature and goat-anti mouse-HRP (1:1000) (Abcam) for 1 hour, room temperature. Samples were incubated with 3, 3', 5, 5'-Tetramethylbenzidine (TMB) substrate (Sigma) for 20 minutes, room temperature and reaction stopped following addition of 1 M H₂S0₄. Relative levels of rScpA binding were quantified as A₄₅₀ values.