Quantifying Anti-Scg3 and Aflibercept Production

Human embryonic kidney 293 cells (HEK293) were seeded on 6-well plates at 2 × 10⁵ cells/well in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1x GlutaMAX (Thermo Fisher) and 1% penicillin/streptomycin (Gibco) and incubated at 37 °C overnight in a humidified atmosphere with 5% CO₂. The cells were transduced with AAV-anti-Scg3Fab, AAV-aflibercept or AAV-mCherry at 5 × 10⁶ vg/mL. The medium was replaced with a serum-free 293SFM II medium (Thermo Fisher) the following day. Five days post-transduction, the conditioned medium was collected and concentrated using an Amicon^® Ultra-4 Centrifugal Filter Unit (UFC801008, Millipore Sigma, St. Louis, MO, USA). An enzyme-linked immunosorbent assay (ELISA) was performed with pre-immobilized Scg3 (5 μg/mL, 100 μL/well, Sino Biological, Wayne, PA, USA), recombinant human VEGF (VEGF, 5 μg/mL, R&D Systems, Minneapolis, MN, USA) or bovine serum albumin (BSA, Sigma). Bound anti-Scg3 mAb and aflibercept were detected with biotin-conjugated anti-FLAG M2 mAb and horseradish peroxidase (HRP)-conjugated streptavidin (Sigma), followed by a colorimetric assay [27 (link)].