Biotinylated goat anti human vegf

PanVEGF capture antibody (1 μg ml⁻¹) (Duoset VEGF ELISA DY-293; R&D Systems, Minneapolis, MN, USA) was incubated overnight at room temperature. The plates were blocked (Superblock; Thermo Scientific) and serial dilutions of recombinant human (rh)VEGF₁₆₅ or rhVEGF₁₆₅b standards (ranging from 4 ng ml⁻¹ to 16.25 pg ml⁻¹) were added, incubated alongside sample lysates, typically diluted 1 : 10. The plate was incubated for 1 h at 37 °C with shaking, washed and incubated with 100 μl per well of either biotinylated goat anti-human VEGF (0.1 μg ml⁻¹; R&D Systems) or mouse anti-human VEGF₁₆₅b (0.25 μg ml⁻¹) for a further 1 h at 37 °C. After washing, 100 μl per well of horseradish peroxidase (HRP)-conjugated streptavidin (1 : 200; R&D Systems) was added and plates were left at room temperature for 20 min.
The plates were washed and colour change was induced with substrate A and B (DY-999; R&D Systems) for 20 min under light protection. The reaction was stopped by addition of 100 μl per well of 1 M H₂SO₄ and the absorbance was read immediately in an ELISA plate reader (Dynex Technologies Opsys MR system plate reader) at 450 nm with a control reading at 570 nm. Revelation Quicklink 4.25 software (Dynex Technologies, Chantilly, VA, USA) was also used to calculate a standard curve from mean absorbance values of standards enabling the estimation of VEGF concentration for each sample.