Guanylate Cyclase Activator 2A (GUCA2A), also known as guanylin, is a peptide hormone secreted by intestinal epithelial cells. It activates membrane-bound guanylate cyclase-C (GC-C) receptor, stimulating cyclic guanosine monophosphate (cGMP) production. This regulates electrolyte and fluid homeostasis, particularly in the gut, influencing chloride secretion and reducing water absorption, which impacts stool consistency. GUCA2A is critical in defending against enteric pathogens and maintaining intestinal barrier integrity. Dysregulation links to diarrheal diseases, constipation, or colorectal cancer. Its expression is modulated by microbial metabolites, dietary factors, and inflammatory signals.Besides,Natriuretic Peptide Receptor 3 (NPR3) has been identified as an interactor of GUCA2A, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human GUCA2A and recombinant rat NPR3. Briefly, GUCA2A was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to NPR3-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-GUCA2A 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 GUCA2A and recombinant rat NPR3 was shown in Figure 1, the EC50?for this effect is 0.092ug/mL.