Heme Oxygenase 1 (HO1) is a stress-inducible rate-limiting enzyme in heme catabolism, catalyzing heme degradation into carbon monoxide, biliverdin and free iron. As a critical cytoprotective protein, it is upregulated by oxidative stress, inflammation and hypoxia, exerting anti-oxidative, anti-inflammatory and anti-apoptotic effects by scavenging reactive oxygen species and regulating iron homeostasis. HO1 is widely expressed in the liver, spleen and vascular tissues, and its dysregulation is associated with cardiovascular diseases, neurodegeneration, cancer and metabolic disorders, making it a promising therapeutic target for oxidative stress-related pathologies. It also modulates immune responses and tissue repair by regulating macrophage polarization and endothelial cell function. HO1 and NQO1 are co-induced by Nrf2, forming a synergistic antioxidant defense network to enhance cellular stress resistance. Briefly, NQO1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to HO1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-NQO1 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. Measured by its binding ability in a functional ELISA. When Recombinant rat HO1 is lmmobilized at 2 µg/mL(100 µLwell), the concentration of NQO1 that produces 50% optimal bindingresponse is found to be approximately 0.024 µg/mL.