Quantikine elisa human vegf immunoassay

To analyze the effect of rhTGFβ on VEGF production, the collected culture supernatant was analyzed using a commercial ELISA kit (Quantikine® ELISA Human VEGF Immunoassay, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. In brief, 200 μl of standard or culture supernatant was added to a primary antibody-coated 96-well plate and incubated for 2 h at room temperature (RT). The wells were then washed three times with wash buffer, and the samples were incubated with 200 μl of horseradish peroxidase-conjugated secondary antibody for 2 h at RT. After three washes, 200 μl of tetramethylbenzidine solution was added to the wells and the samples were incubated at RT. After 20 min, the reaction was stopped by adding sulfuric acid solution, and the absorption was measured using an ELISA reader at 450 nm with lambda correction at 570 nm.

ELISA was performed on conditioned media for validation of the MS data on vascular endothelial growth factor A (VEGF-A; Quantikine® ELISA Human VEGF Immunoassay, R&D Systems™, DVE00), angiopoietin-like 4 (ANGPTL4; DuoSet® ELISA Development system Human Angiopoietin-like 4, R&D Systems™, DY3485), and cathepsin B (CTSB; DuoSet® ELISA Development system Human Total Cathepsin B, R&D Systems™, DY2176). ELISA analysis was performed after the manufacturer’s protocol, and results were normalized to total protein concentrations.