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 0.37ug/mL.