Heme Oxygenase 1 (HO1), a stress-inducible enzyme belonging to the heme oxygenase family, is widely expressed across tissues and cells. It catalyzes the rate-limiting step of heme degradation into biliverdin, iron, and carbon monoxide, with anti-oxidative, anti-inflammatory, and cytoprotective functions. HO1 plays pivotal roles in maintaining cellular redox homeostasis, mitigating oxidative stress, and regulating immune responses, contributing to tissue repair, cancer progression, and cardiovascular health. HO1 interacts with KEAP1, a cysteine-rich adaptor protein, inhibiting KEAP1-mediated ubiquitination of Nrf2 to activate cytoprotective signaling.Briefly, KEAP1 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-KEAP1 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 HO1 is lmmobilized at 2 µg/mL(100 uLwell), the concentration of KEAP1 that produces 50% optimal binding response is found to be approximately 0.019 µg/mL.