Proteinase 3 (PR3) is a serine protease mainly expressed in azurophilic granules of neutrophils. Structurally, it consists of a catalytic domain and a pro - domain. Functionally, PR3 is crucial for neutrophil - mediated immune responses. It can cleave various extracellular matrix proteins and bioactive peptides, playing roles in processes like neutrophil extracellular trap (NET) formation and degradation of pathogens. In diseases, abnormal PR3 activity is associated with autoimmune disorders such as granulomatosis with polyangiitis, where autoantibodies against PR3 are characteristic.The binding of PR3 to IL32 has been reported to enhance the inflammatory response.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human PR3 and recombinant human IL32. Briefly, PR3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to IL32-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-PR3 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 PR3 and recombinant human IL32 was shown in Figure 1, the EC50 for this effect is 0.026ug/mL.