Haptoglobin (Hpt) is an acute-phase plasma glycoprotein primarily produced by hepatocytes. It serves crucial roles in iron homeostasis and antioxidant defense by binding free hemoglobin (Hb) released during hemolysis, forming an irreversible Hpt-Hb complex. This complex is rapidly cleared via macrophage CD163 receptors, preventing Hb-induced oxidative tissue damage and iron loss. As a sensitive acute-phase reactant, Hpt levels elevate markedly during inflammation, infection, or tissue injury, making it a valuable clinical marker. Notably, Hpt can also bind myoglobin (MYO) - though with lower affinity than Hb - potentially alleviating MYO-mediated oxidative stress in rhabdomyolysis or muscle injury.Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat Hpt and recombinant bovine MYO . Briefly, biotin-linked Hpt were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to MYO-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 rat Hpt and recombinant bovine MYO was shown in Figure 1, the EC50 for this effect is 0.57 µg/mL.