Enzyme-linked immunosorbent assay (ELISA)

To analyze interactions between P. aeruginosa OMPs and laminin-111, laminin-111 at concentrations of 0.078 nM–5 nM were coated in 96-well PolySorp plates (Thermo Fisher Scientific, Waltham, MA) in coating buffer (100 mM Tris-HCl, pH 9.0) for overnight at 4 °C. Following a standard ELISA protocol, ligands (recombinant bacterial proteins; 50 nM-100 nM) were added in PBS-0.05% Tween-20 with 1% fish gelatin and bound bacterial proteins were detected using HRP-conjugated anti-His pAbs at dilution 1:5,000. Absorbance was read at 450 nm. The IgD-binding region of Moraxella catarrhalis IgD binding protein (MID 962–1200), which does not bind laminin, was included as a negative control¹⁸. For heparin inhibition, 2.5 nM laminin-111 was coated in microtiter plates and ligands were added together with heparin at 0.1–1000 μg/ml. For analysis of LG domains (1–3, 4 or 5), 0.125–8 μg/ml of the recombinant LG were coated as above. Bound ligands were detected using rabbit pAbs directed to each bacterial protein analysed, and HRP-conjugated swine anti-rabbit pAbs.