Human vegf165

96-well EIA/RIA 3590 plates (Corning Costar) were coated with human VEGF165 (Sino Biological) followed by blocking, before titrated amounts of the anti-VEGF biologics were added to the plates as previously described. Next, 100 μl of recombinant hFcRn-GST was added at a final concentration of 1 μg/mL diluted in S/PBS/T pH 5.5 (100 mM phosphate buffer, 0.15 M NaCl, 4% skimmed milk, 0.05% Tween 20) or S/PBS/T pH 7.4 and incubated for one hour at RT on a shaker^{45 (link)}. After washing with either pH 5.5 or pH 7.4 PBS/T, horse radish peroxidase-conjugated anti-GST (Rockland Immunochemicals Inc) diluted 1:8000 in either pH 5.5 or pH 7.4 PBS/T was added and incubated for one hour at RT on a shaker. After washing as above, the bound receptor was visualized by adding 100 μl tetramethylbenzidine substrate (Calbiochem) followed by adding 100 μl 1 M HCl to stop the reaction. The absorbance was measured at 450 nm using a Sunrise spectrophotometer (Tecan Group Ltd.).

96-well EIA/RIA 3590 plates (Corning Costar) were coated with 100 μl 0.5 µg/mL human VEGF165 (Sino Biological) and incubated over night at 4 °C. The plates were blocked for 2 hours with 250 μl 4% skimmed milk powder (S) (Sigma-Aldrich) dissolved in PBS (Sigma-Aldrich) (S/PBS), followed by washing four times with PBS containing 0.05% Tween20 (T) (Sigma-Aldrich). Next, 100 μl titrated amounts (1000–0.5 ng for ranibizumab and 2000–0.9 ng for aflibercept and bevacizumab) of the anti-VEGF biologics diluted in S/PBS/T were added to the plates and incubated at room temperature (RT) for one hour on a shaker. After washing as previously described, 100 μl alkaline phosphatase (ALP)-conjugated goat anti-hFc Ab (Sigma-Aldrich) or ALP-conjugated anti-hKLC diluted to 1 μg/mL in S/PBS/T was added and incubated for 1 hour on a shaker. Following washing, the bound proteins were visualized by adding 100 μl ALP substrate (1 mg/mL) dissolved in diethanolamine buffer. The absorbance was measured at 405 nm using a Sunrise spectrophotometer (Tecan Group Ltd.).

A Biacore T200 (GE Healthcare) was used for measurements by immobilizing human VEGF165 (Sino Biological) (~300 resonance units (RU)) to CM5 sensor chips using amine-coupling as described by the manufacturer. The coupling was performed by injecting 5 µg/mL human VEGF165 dissolved in 10 mM sodium acetate pH 4.5 and using the amine coupling kit (GE Healthcare). HBS-P+ (0.01 M HEPES, 0.15 M NaCl, 0.005% surfactant P20, pH 7.4) was used as both running and dilution buffer. The measurements were performed by injecting 800 mM ranibizumab or 100 mM aflibercept and bevacizumab over the immobilized VEGF165 at a flow rate of 30 µl/min. Glycine at pH 1.5 (GE Healthcare) was used for regeneration of the CM5 chip between consecutive sample measurements. The sensorgrams were zero-adjusted and the individual injections normalized using the BIAevaluation software version 4.1 (GE Healthcare).