DGKe (Diacylglycerol kinase epsilon) is an enzyme that mainly involved in the regeneration of phosphatidylinositol (PI) from diacylglycerol in the PI-cycle during cell signal transduction. PDHA1 (Pyruvate dehydrogenase E1 component subunit alpha) has been proven to be an interactor of DGKe through Affinity Capture-MS. Thus a binding ELISA assay was conducted to detect the interaction of recombinant human DGKe and recombinant human PDHA. Briefly, DGKe were diluted serially in PBS, with 0.01%BSA (pH 7.4). Duplicate samples of 100uL DGKe were then transferred to PDHA-coated microtiter wells and incubated for 2h at 37°C. Wells were washed with PBST and incubated for 1h with anti-DGKe pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37°C. Finally, add 50μL stop solution to the wells and read at 450nm immediately. The binding activity of DGKe and PDHA was shown in Figure 1, and this effect was in a dose dependent manner.