Calpain 1 (CAPN1),a calcium-dependent cysteine protease, is a member of the calpain family. CAPN1 is primarily found in muscle tissue, particularly in skeletal muscle, where it is involved in the regulation of muscle contraction and relaxation. It is involved in various physiological and pathological processes, such as apoptosis, cell signal transduction and cytoskeleton reconstruction. It is reported that the binding of BID and CAPN1 plays an important role in the process of apoptosis. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human CAPN1 and recombinant human BID. Briefly, CAPN1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to BID-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CAPN1 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. The binding activity of recombinant human CAPN1 and recombinant human BID was shown in Figure 1, the EC50 for this effect is 0.21 ug/mL.