Activating transcription factor 3 (ATF3) is a stress-induced transcription factor that plays vital roles in modulating metabolism, immunity and oncogenesis. ATF3 acts as a hub of the cellular adaptive-response network. Multiple extracellular signals, such as endoplasmic reticulum (ER) stress, cytokines, chemokines, and LPS, are connected to ATF3 induction. Proto-oncogene c-Fos can bind to ATF3 to regulate signal transduction, cell proliferation and differentiation. Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse ATF3 and recombinant human FOS. Briefly, ATF3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FOS-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ATF3 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 ATF3 and recombinant human FOS was shown in Figure 1, the EC50?for this effect is 0.13 ug/mL.