Kallistatin (KAL), a member of serpin family, can Inhibit human amidolytic and kininogenase activities of tissue kallikrein. KAL is widely distributed in a variety of organs and body fluids and has biological effects such as anti-inflammatory, antioxidant stress, anti-angiogenesis and anti-tumor. Besides, Kallikrein 2 (KLK2) has been identified as an interactor of KAL, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human KAL and recombinant human KLK2. Briefly, KLK2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to KAL-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-KLK2 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 450/630 nm immediately. When recombinant human KAL is immobilized at 2 ug/mL (100 uL/well), the concentration of KLK2 that produces 50% optimal binding response is found to be approximately 0.49 ug/mL.