Quantification of VEGF by ELISA (serum and ascites)

The analysis of our study was focused on the most important pro-angiogenic factor VEGF-A (splice variant 165). In order to quantify the secreted amount of VEGF, a quantitative VEGF immunoassay was performed according to the manufacturer’s protocol (R&D Systems, Minneapolis, USA). Briefly, 100 μl of control, sample or standard was added to 100 μl assay diluent. After 2 h incubation at room temperature, the samples were washed three times. Then 200 μl VEGF conjugate was added for 2 h and the samples were washed again. After incubation with 200 μl substrate solution for 25 min, 50 μl stop solution was added and the optical density was measured at 450 nm (Elisareader Sunrise, Tecan, Männedorf, Switzerland).