Vascular Endothelial Growth Factor C (VEGFC) is a key lymphangiogenic factor that primarily binds to VEGFR-3 (FLT4), promoting lymphatic endothelial cell proliferation, migration, and survival. It also activates VEGFR-2 (KDR) upon proteolytic processing, contributing to angiogenesis. VEGFC is essential for embryonic lymphatic development and adult lymphatic remodeling.Besides,Vascular Endothelial Growth Factor D (VEGFD) has been identified as an interactor of VEGFC, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant bovine VEGFC and recombinant bovine VEGFD . Briefly, biotin-linked VEGFC were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to VEGFD-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μl stop solution to the wells and read at 450nm immediately. The binding activity of recombinant bovine VEGFC and recombinant bovine VEGFD was shown in Figure 1, the EC50?for this effect is 0.33ug/mL.