Aldolase B, Fructose-Bisphosphate (ALDOB)?is a glycolytic enzyme that catalyzes the reversible cleavage of fructose-1,6-bisphosphate (F1,6BP) into glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Predominantly expressed in the liver, kidney, and small intestine, ALDOB plays a key role in gluconeogenesis and glycolysis.Besides,PFKP has been identified as an interactor of ALDOB, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human ALDOB and recombinant human PFKP. Briefly, ALDOB was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to PFKP-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ALDOB 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 ALDOB and recombinant human PFKP was shown in Figure 1, the EC50?for this effect is 0.058ug/mL.