IL-35, also known as Interleukin-35, is a heterodimeric cytokine composed of two subunits, p35 and Epstein-Barr virus-induced gene 3 (EBI3). It belongs to the IL-12 family of cytokines and is primarily associated with the regulation of immune responses. IL-35 is produced by regulatory T cells (Tregs) and has been implicated in the suppression of immune responses, playing a role in maintaining immune tolerance and preventing autoimmune diseases. It is also produced by B regulatory cells and can contribute to the suppression of immune reactions in certain contexts.Besides,Interleukin 23 Subunit Alpha (IL23a) has been identified as an interactor of IL-35, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse IL-35 and recombinant human IL23a. Briefly, IL-35 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to IL23a-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IL-35 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. The binding activity of recombinant mouse IL-35 and recombinant human IL23a was shown in Figure 1, the EC50?for this effect is 0.049ug/mL.