Fibroblast Growth Factor 4 (FGF4) is a secreted signaling protein belonging to the fibroblast growth factor family, which plays crucial roles in embryonic development, cell proliferation, differentiation, and migration. It is particularly important during limb formation, mesoderm patterning, and stem cell maintenance. FGF4 exerts its biological functions by binding and activating specific FGF receptors (FGFRs) with high affinity. FGF4 specifically binds to Fibroblast Growth Factor Receptor 2 (FGFR2), particularly the IIIc isoform, triggering receptor dimerization, autophosphorylation, and subsequent activation of downstream signaling pathways including MAPK and PI3K-AKT.To detect the activity of recombinant FGF4 , a functional ELISA assay was performed to evaluate the interaction between recombinant human FGF4 and recombinant rat FGFR2.Briefly, FGF4 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FGFR2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-FGF4 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 FGF4 and recombinant rat FGFR2 was shown in Figure 1, the EC50?for this effect is 0.612 μg/mL.