Heme Oxygenase 1 (HO1) is a stressinducible, cytoprotective enzyme that catalyzes the ratelimiting step in heme catabolism. It degrades prooxidant heme into equimolar amounts of biliverdin, free iron, and carbon monoxide (CO). HO1 is strongly upregulated under oxidative stress, inflammation, hypoxia, and cytotoxic conditions, exerting antioxidative, antiapoptotic, and antiinflammatory effects in various tissues. It plays essential roles in vascular homeostasis, cellular defense, and protection against tissue injury. Dysregulation of HO1 is associated with inflammatory diseases, cardiovascular disorders, and cancer progression. HO1 physically interacts with AIMP1 to modulate cellular stress responses and regulate protein homeostasis under harmful stimuli.Briefly, AIMP1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to HO-1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-AIMP1 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 cattle HO-1 is lmmobilized at 2 µg/mL(100 µLwell), the concentration of AIMP1 that produces 50% optimal bindingresponse is found to be approximately 0.072 µg/mL.