Heme Oxygenase 1 (HO1) is an enzyme that catalyzes the rate-limiting step in heme degradation, converting heme into biliverdin, carbon monoxide (CO), and free iron. Expressed primarily in the spleen and liver, HO1 is induced by oxidative stress, inflammation, and hypoxia. It plays critical roles in cellular protection by mitigating oxidative damage, modulating inflammation, and promoting iron recycling. HO1's products, particularly CO and biliverdin (later reduced to bilirubin), exhibit antioxidant, anti-apoptotic, and vasodilatory properties. Its activity is tightly regulated and linked to pathophysiological processes, including ischemia-reperfusion injury, atherosclerosis, and neurodegenerative diseases.Biliverdin Reductase B (BLVRB) collaborates with HO1 , which rapidly reduces biliverdin to bilirubin. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant bovine HO1 and recombinant human BLVRB . Briefly, biotin-linked HO1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to BLVRB-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 recombinant bovine HO1 and recombinant human BLVRB was shown in Figure 1, and this effect was in a dose dependent manner.