Monoacylglycerol lipase (MGL) is a crucial serine hydrolase primarily responsible for the hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG), thereby terminating its signaling. It plays a central role in regulating endocannabinoid tone, metabolism, and various physiological processes, including pain sensation, inflammation, and neuronal excitability. Dysregulation of MGL activity is implicated in numerous neurological disorders and cancer. While MGL and Sonic Hedgehog (SHH) operate in distinct signaling pathways, emerging research suggests a potential functional intersection in specific contexts like tumorigenesis, where MGL-derived lipid signals might indirectly influence the SHH pathway.Besides,SHH has been identified as an interactor of MGL, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human MGL and recombinant mouse SHH. Briefly, MGL was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to SHH-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-MGL 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 MGL and recombinant mouse SHH was shown in Figure 1, the EC50 for this effect is 0.43498µg/mL..