Granzyme B (GZMB) is a serine protease predominantly secreted by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It serves as a central effector molecule in the granule exocytosis pathway, which is essential for cell-mediated elimination of virus-infected and malignant cells. Upon target cell recognition, CTLs and NK cells release GZMB together with perforin. Perforin facilitates the entry of GZMB into the target cell by forming pores in the membrane, where GZMB then initiates apoptotic signaling. In addition to its well-established role in apoptosis, GZMB also contributes to immune regulation and tissue remodeling through involvement in inflammatory responses and extracellular matrix degradation. A key mechanism of its pro-apoptotic function involves the direct cleavage of Bid, generating truncated Bid (tBid), which potently activates the mitochondrial apoptosis pathway.To detect the activity of recombinant GZMB, a functional ELISA assay was performed to evaluate the interaction between recombinant rat GZMB and recombinant human Bid. Briefly, biotin-linked GZMB were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to Bid-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μl stop solution to the wells and read at 450nm immediately. The binding activity of GZMB and Bid was shown in Figure 1, the EC50?for this effect is 0.16μg/mL.