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. Myostatin (MSTN) 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 human MSTN. Briefly, FNDC5 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to MSTN-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 human MSTN was shown in Figure 1, the EC50 for this effect is 0.03 ug/mL.