Haptoglobin (Hpt) a secreted acute-phase glycoprotein mainly produced by hepatocytes. Composed of α and β polypeptide chains connected by disulfide bonds, Hpt circulates widely in plasma. Its canonical function is sequestering free hemoglobin from hemolysis to block oxidative stress and avoid kidney damage. Moreover, Hpt participates in immune response regulation, inflammation control and lipid metabolism. Its expression rises rapidly under infection or tissue injury, making it a routine clinical inflammatory indicator. Different Hpt isoforms differ in antioxidant capacity and are closely linked to metabolic and cardiovascular illnesses. Hpt interacts with PANX1 to regulate channel function and facilitate intercellular inflammatory signal communication. Thus a functional ELISA assay was conducted to detect the interaction of recombinant human Hpt and recombinant human PANX1. Briefly, Hpt was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to PANX1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-Hpt 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. The binding activity of recombinant human Hpt and recombinant human PANX1 was shown in Figure 1, the EC50?for this effect is 0.210 μg/mL.