Anti vitronectin

Ninety six-well plate were coated overnight at 4°C with 100 μl/well containing 1.6 × 10⁸ spores or 1 μg of BSA in PBS (as a control). Then, the wells were washed five times with wash PBS containing 0.05% Tween 20 (PBST), blocked with 100 μl of PBS containing 2%-BSA and 0.05%-Tween 20 for 1 h at 37°C and subsequently incubated with 50 μl containing increased quantities of fibronectin or vitronectin (i.e., 0, 1, 10, 100, and 1000 nM) for 1 h at 37°C. Wells were then rinsed 5 times with 150 μl PBST prior to incubation with 50 μl of anti-fibronectin or anti-vitronectin (Santa Cruz Biotechnology, USA) at a 1:1000 dilution in PBST containing 1% BSA for 1 h at 37°C. Then, wells were rinsed 5 times with 150 μl of PBST and incubated for 1 h at 37°C with 50 μl of 1%-BSA in PBST containing a 1:5000 dilution of anti-rabbit (Rockland Inc. USA). After 5 washings with 150 μl of PBST, wells were rinsed once with 150 μl of 50 mM carbonate buffer (pH 9.6) prior to incubation with 50 μl of reaction buffer (17 mM citric acid, 65 mM potassium phosphate, 0.15%-hydrogen peroxide, 0.4%-θ-phenylenediamine for 20 min at room temperature. Reaction was stopped with 25 μl of 4.5N sulfuric acid. Binding was quantified by colorimetric detection at 450 nm in a 96-well plate reader (Infinite F50, Tecan, USA).