Interleukin-4 (IL4) is a pivotal pleiotropic cytokine primarily produced by activated T helper 2 (Th2) cells, mast cells, and basophils. It is a central regulator of the adaptive immune response, driving the differentiation of naive T cells into the Th2 lineage. IL4 is renowned for its role in promoting humoral immunity by stimulating B-cell proliferation, class-switching to IgE and IgG1, and antibody production. Furthermore, it induces alternative activation of macrophages, contributing to anti-inflammatory responses and tissue repair. The biological functions of IL4 are initiated by its specific binding to the membrane-bound Interleukin-4 Receptor (IL4R), forming a signaling complex that activates the JAK-STAT pathway.To detect the activity of recombinant IL4 , a functional ELISA assay was performed to evaluate the interaction between recombinant chicken IL4 and recombinant human IL4R.Briefly, IL4R was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to IL4-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IL4R 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.Measured by its binding ability in a functional ELISA. When Recombinant IL4 is lmmobilized at 2 µg/mL(100 uLwell), the concentration of IL4R that produces 50% optimal bindingresponse is found to be approximately 0.256 µg/mL.