Fibronectin type III domain-containing protein 5, the precursor of irisin, is a protein that is encoded by the FNDC5 gene. FNDC5 is a 12 kDa glycosylated polypeptide hormone that regulate glycolipid metabolism, cardiovascular homeostasis metabolism, stem cell differentiation, and neuronal development. Brain Derived Neurotrophic Factor (BDNF) has been identified as an interactor of FNDC5, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human FNDC5 and recombinant dog BDNF. Briefly, FNDC5 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to BDNF-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-FNDC5 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 FNDC5 and recombinant dog BDNF was shown in Figure 1, the EC50 for this effect is 1.64 ug/mL.