Interferon Alpha 5 (IFNa5), a member of the type I interferon family, is a cytokine primarily expressed by virus-infected cells and immune cells?. It binds to the interferon-α/β receptor (IFNAR) to activate JAK-STAT signaling, inducing expression of interferon-stimulated genes (ISGs). This triggers antiviral, antiproliferative, and immunomodulatory responses, inhibiting viral replication and enhancing immune cell activity. IFNa5 exhibits therapeutic potential in treating viral infections (e.g., hepatitis B/C) and cancers. Its activity is tightly regulated to balance immune defense and avoid excessive inflammation or autoimmunity.Besides,Interferon Gamma Receptor 1 (IFNgR1) has been identified as an interactor of IFNa5, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse IFNa5 and recombinant human IFNgR1 . Briefly, biotin-linked IFNa5 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to IFNgR1-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 IFNa5 and IFNgR1 was shown in Figure 1, the EC50?for this effect is 0.307ug/mL.