Epithelial Neutrophil Activating Peptide 78 (ENA78), encoded by the CXCL5 gene, is a key CXC chemokine primarily secreted by epithelial cells, macrophages, and neutrophils. Initially identified for its potent neutrophil-activating and chemotactic activities, it plays critical roles in innate immunity, inflammation, and tissue repair by recruiting and activating neutrophils at sites of infection or injury. ENA78 expression is upregulated in inflammatory conditions like sepsis, asthma, and rheumatoid arthritis, as well as in various cancers where it promotes tumor angiogenesis and metastasis. Its biological functions are mediated through binding to the CXCR2 receptor on target cells, triggering downstream signaling cascades. ENA78 and CXCL2 (GROβ) are homologous CXC chemokines that share the CXCR2 receptor, enabling functional redundancy in neutrophil recruitment.Besides,CXCL2 has been identified as an interactor of ENA78, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant moiuse ENA78 and recombinant human CXCL2. Briefly, ENA78 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CXCL2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ENA78 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 ENA78 and recombinant human CXCL2 was shown in Figure 1, the EC50?for this effect is 1.06326μg/mL.