Neuregulin 4 (NRG4) is a member of the neuregulin family of growth and differentiation factors, structurally related to epidermal growth factor (EGF). It is primarily expressed in brown adipose tissue and plays critical roles in regulating energy metabolism, thermogenesis, and adipocyte function. NRG4 acts as a ligand for ErbB receptor tyrosine kinases, especially ErbB4, activating downstream signaling pathways involved in cell survival, proliferation, and metabolic homeostasis. Emerging evidence links NRG4 dysfunction to obesity, diabetes, and metabolic disorders, highlighting its potential as a biomarker and therapeutic target for metabolic diseases. Unlike NRG1, NRG4 does not bind directly; both activate ErbB receptors but via distinct binding preferences and tissue distributions.To detect the activity of recombinant NRG4 , a functional ELISA assay was performed to evaluate the interaction between recombinant human NRG4 and recombinant human NRG1. Briefly, NRG4 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to NRG1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-NRG4 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 mouse NRG4 and recombinant human NRG1 was shown in Figure 1, the EC50?for this effect is 0.149 μg/mL.