Hrp substrate

The availability of
FN adsorbed on polymer surfaces as well as that of the RGD and synergy
domains was determined by ELISA. Samples were blocked for 30 min with
1% w/v BSA (Sigma-Aldrich). To determine the availability of FN, we
incubated rabbit polyclonal anti-FN antibody (Sigma-Aldrich, 1:10 000)
for 1 h, followed by a 1 h incubation with biotinylated horse antirabbit
secondary antibody (Vectorlabs, 1:10 000), both at room temperature
(RT). Samples were then incubated with HRP-streptavidin (R&D Systems)
for 20 min, washed, and incubated with HRP substrate (R&D Systems)
for 20 min. After stopping the reaction, using the stop solution,
absorbance was measured at 450 (maximal absorbance of the tag) and
540 nm (blank control, to determine background absorbance). To assess
the availability of cell-binding domains, we incubated samples at
RT with monoclonal mouse primary antibody (mAb1937, 1:20,000 in 1%
BSA or HFN 7.1 antibody for the synergy or RGD domain, respectively).
The samples were then washed with 0.5% Tween 20 and incubated with
goat antimouse HRP-tagged secondary antibody (1:10 000 in 1%
BSA solution) for 1 h (RT). After washing with 0.5% Tween 20, samples
were incubated with HRP substrate solution in the absence of light
for 20 min. The reaction was terminated with stop solution (R&D
Systems) and absorbance was measured.