Figure 6. Sandwich assay for looking at receptor-cytokine interactions. (i) Sandwich biosensor-binding analysis of IL5Rα variants. (A) Anti-V5 tag monoclonal antibody is covalently immobilized to the carboxymethyldextran (CM-dextran) matrix on sensor chip gold surface. The cell-free culture supernatant containing a carboxy-terminally V5-tagged receptor (e.g., IL5Rα) is injected across all the flow cells. Anti-V5 tag monoclonal antibody can capture IL5Rα selectively, whereas few nonspecific components are bound to the 17b control surface. (B) IL5Rα is captured on the surface via tight interaction of V5 tag and the antibody, providing a configuration similar to that of cell-surface receptors. (C) The captured soluble receptor is then amenable to binding analyses with ligands such as IL5 or monoclonal antibodies. (ii) Sandwich biosensor-binding analysis of IL5 variants. (A) Anti-IL5 nonneutralizing antibody 24G9 is covalently immobilized to the dextran matrix on sensor chip gold surface. The cell-free culture supernatant containing IL5 is injected onto all the flow cells. The antibody can capture wild-type IL5 as well as IL5 mutational variants. (B) IL5 is captured on the surface via tight interaction of the antibody, whereas other cell-culture components are removed after injection of running buffer. (C) Various concentrations of soluble IL5Rα are then injected onto an IL5-captured surface, and the kinetics of interaction are examined.