Peptidyl Arginine Deiminase Type II (PADI2) is a calcium-dependent enzyme that catalyzes the post-translational deimination of arginine residues to citrulline, playing key roles in epigenetic regulation, immune response, and diseases like rheumatoid arthritis and multiple sclerosis. PADI2 interacts with Carbonic Anhydrase I (CA1), potentially modulating cellular pH balance and metabolic processes through their functional interplay. Thus a functional ELISA assay was conducted to detect the interaction of recombinant human PADI2 and recombinant human CA1. Briefly, PADI2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CA1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-PADI2 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 PADI2 and recombinant human CA1 was shown in Figure 1, the EC50 for this effect is 0.054ug/mL.