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.