Alpha-1-Antitrypsin (a1AT), a major serine protease inhibitor synthesized primarily in the liver, is crucial for protecting tissues from excessive proteolytic damage.?Circulating in human plasma, it targets and inhibits a range of proteases—including neutrophil elastase, trypsin, and chymotrypsin—which, if left unregulated, can degrade essential structural proteins. Beyond its protective function, A1AT is involved in modulating inflammatory responses and maintaining pulmonary homeostasis; deficiencies in A1AT are associated with conditions such as emphysema and liver disorders.Notably, a1AT directly binds to Neutrophil Elastase (NE), a potent protease released by activated neutrophils, irreversibly inhibiting NE’s tissue-damaging activity.To detect the activity of recombinant a1AT , a functional ELISA assay was performed to evaluate the interaction between recombinant human a1AT and recombinant human NE.Briefly, a1AT was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to NE-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-a1AT 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 a1AT and recombinant human NE was shown in Figure 1, the EC50?for this effect is 0.07 μg/mL.