Interleukin?28B (IL28B), also designated IFN?λ3, belongs to class?II cytokine superfamily and exerts antiviral, antitumor and immunomodulatory bioactivities. It primarily binds IFNLR1 with high affinity and subsequently recruits IL10RB to assemble a functional heterodimeric receptor complex via protein?protein interaction. This binding event triggers downstream JAK?STAT signaling cascades, inducing interferon?stimulated genes and mediating mucosal immune defense against invading pathogens.
Thus a functional ELISA assay was conducted to detect the interaction of recombinant IL28B and recombinant IFNLR1. Briefly, IL28B was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to IFNLR1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IL28B 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 IL28B and recombinant IFNLR1 was shown in Figure 1, the EC50?for this effect is 0.132μg/mL.