Human vegf a165

A MSD high bind 96-well plate was coated with 2 μg/ml human VEGF-A₁₆₅ (purified as described above), VEGF-A₁₂₁, VEGF-B₁₆₇, VEGF-C, VEGF-D, or mouse VEGF-164 (all from R&D Systems) in PBS, and the sealed plates were incubated overnight at 4 °C. The following day the plates were washed with Tris wash buffer (MSD) and blocked with 3% BSA in PBS for 1 h at room temperature. Plates were washed as described above before the addition of standard concentrations of VEGF dual dAb diluted in 0.1% BSA in PBS and incubated at room temperature for 2 h. Plates were washed again before the addition of the detection reagent (in-house sulfo-TAG-labeled goat anti-human IgG, Fc-specific; Sigma) at 1 μg/ml in 1% BSA in PBS and incubated at room temperature for 1 h. Plates were washed, and 2× Read Buffer T (MSD) was added before being read on a SECTOR 6000 MSD imager. GraphPad Prism was used to fit curves using a sigmoidal dose response (variable slope) model and to calculate the EC₅₀ values, (with S.D. calculated over the mean) from n = 3 experiments.