Interleukin 12 (IL12) is a heterodimeric cytokine composed of p35 (IL12A) and p40 (IL12B) subunits, primarily produced by dendritic cells, macrophages, and B cells. It plays a critical role in bridging innate and adaptive immunity by promoting Th1 differentiation, enhancing IFN-γ production, and stimulating NK and T cell cytotoxicity. IL12 is pivotal in anti-tumor and anti-infectious responses but dysregulation can contribute to autoimmunity.IL12 binds IL12Rb1 (a subunit of IL12 receptor) with high affinity, initiating JAK-STAT signaling.Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human IL12 and recombinant mouse IL12Rb1 .Briefly, biotin-linked IL12 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to IL12Rb1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μl stop solution to the wells and read at 450nm immediately. The binding activity of IL12 and IL12Rb1 was shown in Figure 1, the EC50?for this effect is 0.23ug/mL.