Trefoil Factor 3 (TFF3) is a small secretory protein primarily produced by gastrointestinal goblet cells, belonging to the trefoil factor family (TFF) characterized by a conserved trefoil domain. It plays crucial roles in mucosal protection, epithelial repair, and inflammation regulation by resisting protease degradation and promoting cell migration. TFF3 is highly expressed in the intestines and colon, where it stabilizes the mucus layer and facilitates wound healing. Additionally, TFF3 directly binds to FCGBP to form a heteromeric complex that enhances mucosal stability and repair.To detect the activity of recombinant TFF3, a functional ELISA assay was performed to evaluate the interaction between recombinant rat TFF3 and recombinant human FCGBP.Briefly, biotin-linked TFF3 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µl were then transferred to FCGBP-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 TFF3 and FCGBP was shown in Figure 1, the EC50 for this effect is 0.21µg/mL..