Glucocorticoid Receptor (GR) is a member of the steroid receptor superfamily, which includes receptors for other steroid hormones such as estrogens, progestogens, androgens and mineralocorticoids. GR is widely distributed in various types of human cells, especially in liver, muscle, adipose tissue, lung, brain and other organs. GR plays a role in signaling within cells. When glucocorticoids enter cells and bind to GR, GR undergoes conformational changes that activate its transcriptional activity. It has been identified that the binding of Heat Shock 70kDa Protein 4 (HSPA4) to GR plays a key role in glucocorticoid signaling, which not only participates in the stabilization and activation of receptors, but also may affect the effect of drugs. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human GR and recombinant human HSPA4. Briefly, GR was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to HSPA4-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-GR 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 GR and recombinant human HSPA4 was shown in Figure 1, the EC50 for this effect is 5.1 ug/mL.