PRL (prolactin), also known as luteotropin, is a hormone secreted from the pituitary gland and is best known for its role in enabling mammals to produce milk. PRL plays an essential role in metabolism, regulation of the immune system through activating its specific membrane-anchored receptor (PRLR). A functional binding ELISA assay was conducted to detect the interaction of recombinant horse PRL and recombinant human PRLR. Briefly, PRL was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to PRLR-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-PRL 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 horse PRL and recombinant human PRLR was shown in Figure 1, the EC50 for this effect is 0.11 ug/mL.