2.5. Cell-based Reporter Gene Bioassay
The KDR HEK293 cell clone (Cat. CS181401; Promega Corp., Madison, WI, USA) with the VEGF receptor and response element (RE) upstream of luciferase expression was used in the anti-VEGF antibody bioassay. The sample was diluted to a primary concentration of 100 μg/mL in a culture medium of 95% DMEM/high glucose and 5% FBS at a dilution ratio of 1:4 to achieve a concentration of 1.53 ng/mL. The VEGF (Cat. No. 293-VE-50μg; R&D Systems, Minneapolis, MN, USA) was diluted to a concentration of 120 ng/mL in the same culture medium. Equal volumes of gradient dilution sample and VEGF solution were mixed. Then, 50 μL of the mixing solution was added to the white 96-well plate (Cat. 136101, Nunc; Thermo Fisher Scientific) at room temperature for 30 min. We added 50 μL of cells at a density of 1.0 × 106 wells/mL to the plate and incubated for 7 h at 37°C with 5% CO2 to obtain the luciferase activity curves using the Bio-Glo Luciferase Assay System (Cat. No. G7940; Promega).