Galectin 9 (GAL9) is a soluble β-galactoside-binding lectin widely expressed in immune cells and epithelial tissues. It participates in innate and adaptive immune regulation by modulating T cell exhaustion, macrophage polarization and inflammatory responses. GAL9 mediates immune homeostasis under physiological conditions, while its abnormal secretion drives chronic inflammation, autoimmune injury and tumour immune escape. It also controls cell adhesion, apoptosis and cytokine release to shape the local immune microenvironment, becoming a key checkpoint molecule linking inflammation and malignant progression. GAL9 directly binds to HAVCR2 to trigger inhibitory immune signalling. Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse GAL9 and recombinant human HAVCR2. Briefly, GAL9 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to HAVCR2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-GAL9 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 mouse GAL9 and recombinant human HAVCR2 was shown in Figure 1, the EC50?for this effect is 1.139 μg/mL.