Glycosylphosphatidylinositol Specific Phospholipase D1 (GPLD1) is a secreted enzyme that cleaves glycosylphosphatidylinositol (GPI) anchors, releasing GPI-anchored proteins from cell membranes into circulation. Primarily synthesized in the liver, it plays key roles in lipid metabolism, inflammation, and neurogenesis. GPLD1's activity modulates the bioavailability of important GPI-anchored proteins like alkaline phosphatase and folate receptors. Studies link elevated GPLD1 levels to metabolic disorders and aging-related cognitive decline, while its neuroprotective effects are being investigated. The enzyme's structure contains catalytic domains essential for hydrolyzing GPI linkages, making it a potential therapeutic target for metabolic and neurological diseases.Besides,Alkaline Phosphatase, Tissue-nonspecific (ALPL) has been identified as an interactor of GPLD1, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human GPLD1 and recombinant human ALPL. Briefly, GPLD1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ALPL-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-GPLD1 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 GPLD1 and recombinant human ALPL was shown in Figure 1, the EC50?for this effect is 0.042μg/mL.