Uracil Phosphoribosyltransferase (UPRT) is an important enzyme involved in the pyrimidine salvage pathway, which is a critical metabolic pathway for the synthesis of nucleotides in various organisms, including humans. This enzyme plays a key role in the conversion of uracil, a pyrimidine base, into uridine-5'-phosphate (UMP), a precursor for the synthesis of RNA and DNA. Besides, Purine Nucleoside Phosphorylase (PNP) has been identified as an interactor of UPRT, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human UPRT and recombinant mouse PNP. Briefly, UPRT was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to PNP-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-UPRT 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 450/630 nm immediately. The binding activity of recombinant human UPRT and recombinant mouse PNP was shown in Figure 1, the EC50?for this effect is 0.017 ug/mL.