Goat anti mouse hrp conjugate

ELISA assays were carried out using standard protocols68 in 96-well Maxisorb plates (NUNC; Thermo Scientific). Cells were diluted to a 0D₆₀₀ of 0.2 (2 × 10⁸ c.f.u. ml⁻¹), with 50 μl added per well. All the strains were assayed in triplicate. Primary antibody AD6 against HMW34 (link) was used at a starting concentration of 1:200 (NTHi strains C486 and 477) or 1:10,000 (NTHi strain 723) and serially diluted two-fold in 1 × PBS pH 7.9. Secondary antibody (goat anti-mouse HRP conjugate; Sigma-Aldrich) was used at a concentration of 1:10,000. Antibody was detected using TMB single-substrate solution as recommended by the manufacturer (Sigma-Aldrich). The data were plotted as antibody dilution (x axis) versus absorbance at 450 nm (y axis), and data from specific titres in the linear range of the response curve picked for statistical analysis.

Gradient samples were separated on a 4–20% Tris-Glycine acrylamide gel using a BioRad MiniProtean III rig, at a set voltage of 120 V for 60–90 min. Gels were then dismantled and placed within a pre-wetted sandwich of thick blotting paper on top of a pre-cut PVDF membrane (0.45 μm) that had been activated in 100% MeOH for 5 min at RT. Proteins were transferred for 2 hr using a Hoeffer semi-dry transfer rig set at 320 mA. Blotting sandwiches, gels and membranes were thoroughly pre-soaked in transfer buffer (1 x Tris-Glycine pH 8.3, 20% MeOH) prior to assembly. Membranes were removed from transfers, and placed in 20 mL blocking solution (5 % w/v fat-free milk in 1 x Tris-buffered Saline, 0.1% Tween 20 (TBS-T)) and shaken at RT for at least 3–4 hr. Membranes were then washed in TBS-T and placed in 10 mL of blocking solution containing primary antibody (mouse anti-HCV core, C7-50, Thermo Fisher catalogue # MA1-080) diluted 1/1000, at 4°C overnight with gentle shaking. The next day, primary antibody was removed by three washes in 1 x TBS-T at RT for 10 min, followed by incubation with secondary antibody diluted 1/5000 in blocking solution (goat anti-mouse HRP conjugate, SIGMA) for 2 hr at RT. Washing was then repeated prior to visualisation using ECL prime chemiluminescence substrate (GE Healthcare/Amersham), according to the manufacturer’s instructions.