The plate was washed three times with wash buffer and plates were incubated with anti-His secondary antibody (Quiagen,UK) at a dilution of 1∶1000 in 0.05% BSA-PBS pH 7.4 for 1 h.
After washing three times with PBS, 0.5% (v/v) Tween-20 (washing buffer), the plates were incubated with 0.17 μg/ml biotinylated anti-porcine IFN-γ Ab (P2C11, BD Biosciences, Allschwil, Switzerland) in PBS at 4°C O/N.
After two washes with binding buffer, the plates were incubated with I-RGDyK (2 nmol/L) in the presence of various concentrations of ligand ranging from 1 pM to 10 μM for 16 h at room temperature, followed by three washes with binding buffer.
Plates were then washed, polyclonal anti-human GDNF in block and sample buffer was added, and plates were incubated overnight at 4°C.
After 4 hours, Lysis Buffer was added, and plates were incubated an additional 24 hours.
After incubation, the plate was washed as above, and alkaline phosphatase-conjugated goat anti-rabbit IgG, diluted 2000-fold in assay buffer, was applied and plates were incubated for 15 min.
After washing, 0.1 mL/well of biotinylated goat anti-mouse IgA (Southern Biotechnologies; cat# 1040-08) diluted 1 5,000 in assay buffer was added and plates were incubated for 2 hours at RT.
Certified High Bind 96-well plates (Corning) were coated with 0.1 μg/ml of mouse anti-HSP70 monoclonal antibody (Stressgen) diluted in 0.1 m carbonate buffer, pH 9.6, and plates were incubated at 4°C overnight.
Microtitre plates were coated with 50 μl of capture antibody for IL-4 (rat antimouse IL-4 used at 2 μg/ml; Pharmingen, San Diego, CA, USA) or IFN-γ (rat antimouse IFN-γ used at 5 μg/ml; Pharmingen), both antibodies were diluted with 0.5 mol/l carbonate/bicarbonate buffer (pH 9.6), and plates were incubated overnight at 4°C.
