Palmitoyl Protein Thioesterase 1 (PPT1)?is a lysosomal enzyme that removes palmitate groups from S-palmitoylated proteins, playing a crucial role in protein degradation and recycling. Mutations in the?PPT1?gene cause?infantile neuronal ceroid lipofuscinosis (CLN1 disease), a severe neurodegenerative lysosomal storage disorder characterized by vision loss, seizures, and cognitive decline. PPT1's thioesterase activity is essential for breaking down lipid-modified proteins in lysosomes, preventing toxic accumulation. It also influences synaptic function and neuronal survival, linking lipid metabolism to neurodegeneration.Besides,Tripeptidyl Peptidase I (TPP1) has been identified as an interactor of PPT1, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human PPT1 and recombinant mouse TPP1 . Briefly, biotin-linked PPT1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to TPP1-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 PPT1 and TPP1 was shown in Figure 1, the EC50?for this effect is 0.50 μg/mL.