Vascular Endothelial Growth Factor D (VEGFD) is a crucial member of the VEGF family, encoded by the VEGFD gene in humans. It is synthesized as an inactive precursor protein and activated via proteolytic cleavage. VEGFD exerts biological functions primarily through binding to two specific receptors, VEGFR-2 and VEGFR-3, expressed on vascular and lymphatic endothelial cells. These bindings trigger downstream signaling pathways, regulating angiogenesis (new blood vessel formation) and lymphangiogenesis (lymphatic vessel development). Beyond physiological processes like embryonic development and tissue repair, abnormal VEGFD expression is closely linked to pathological conditions, especially cancer. It promotes tumor angiogenesis and lymphatic metastasis, making it a potential therapeutic target for cancer and other angiogenesis-related disorders.To detect the activity of recombinant VEGFD , a functional ELISA assay was performed to evaluate the interaction between recombinant human VEGFD and recombinant rat FGFR1.Briefly, VEGFD was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FGFR1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-VEGFD pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 μL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant human VEGFD and recombinant rat FGFR1 was shown in Figure 1, the EC50?for this effect is 0.12881μg/mL.