Hexokinase 2 (HK2), also referred to as Hexokinase type II or Muscle form hexokinase, is an enzyme that belongs to the hexokinase family. This protein is widely expressed in a variety of tissues, especially in muscle and heart, and is highly expressed in many types of cancer .HK2 is a multifaceted enzyme that is essential for glucose metabolism and has been implicated in a wide range of cellular processes and diseases, including cancer and immune regulation.Besides,Hexokinase 1 (HK1) has been identified as an interactor of HK2, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human HK2 and recombinant human HK1.Briefly, HK2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to HK1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-HK2 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 HK2 and recombinant human HK1 was shown in Figure 1, the EC50?for this effect is 0.12ug/mL.