Figure 1

Soluble interleukin-18 receptor α complex is associated with interleukin-18 and the soluble form of the interleukin-18 receptor β chain. (A) Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the serum interleukin (IL)-18 receptor α (IL-18Rα) complex. Purified H44 monoclonal antibody (mAb) was coupled to a 5-mL HiTrap NHS-activated HP column (GE Healthcare). Pooled human blood serum (120 mL) was applied to this affinity column. The IL-18Rα complex was eluted with elution buffer at a flow rate of 2 mL/minute. Every 1 mL of the elution buffer was collected into a test tube containing 50 mL of neutralization buffer (collected fractions were denoted in order as fractions 1, 2, 3 and so on). A 10-μL aliquot of every fraction (fractions 1 to 8) was treated with the same volume of sample buffer containing 4% SDS (Tris-Glycine SDS Sample Buffer (2×); Invitrogen). Electrophoresis was carried out in the presence of 0.1% SDS, and the gel was stained with Coomassie Brilliant Blue. (B) Western blots of the serum IL-18Rα complex using an antihuman IL-18Rα mAb are shown. Western blot analysis was performed using antihuman IL-18Rα mAb 70625 (R&D Systems, Inc.). Lane 1: Western blot showing 1 μg of rhIL-18Rα/Fc chimera protein (R&D Systems, Inc.). Lane 2: Western blot showing 5 μg of isolated serum IL-18Rα complex. (C) Western blot showing serum IL-18Rα complex using an antihuman IL-18 mAb. Western blot analysis was performed using antihuman IL-18 mAb clone 8 with 5 μg of isolated serum IL-18Rα complex. (D) Western blot showing serum IL-18Rα complex using an antihuman IL-18Rβ mAb. Western blot analysis was performed using antihuman IL-18Rβ mAb 132016 (R&D Systems, Inc.) with 5 μg of isolated serum IL-18Rα complex.