Deoxyribonuclease 1 Like 3 (DNASE1L3) is a mammalian endonuclease predominantly expressed in macrophages and dendritic cells. It plays a critical role in degrading extracellular DNA during programmed cell death (apoptosis), thereby preventing the accumulation of self-DNA and the subsequent initiation of autoimmune responses. Notably, DNASE1L3 is essential for cleaving chromatin within neutrophil extracellular traps (NETs) and circulating cell-free DNA (cfDNA), which is pivotal for maintaining immune tolerance. Besides,Deoxyribonuclease I (DNASE1) has been identified as an interactor of DNASE1L3, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse DNASE1L3 and recombinant human DNASE1 . Briefly, biotin-linked DNASE1L3 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µl were then transferred to DNASE1-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 DNASE1L3 and DNASE1 was shown in Figure 1, the EC50 for this effect is 0.58 µg/mL.