Figure 5

Characteristics of the selected candidate proteins. (A) Specificity analysis by competition assay. The candidates (R10-14 and 15 shown here) at 100 nM were incubated with 1 μM IL-6R, AChBP or IgG. The unbound proteins were captured by immobilized IL-6R and detected using Penta.His HRP. Non-coated beads were used as negative controls and IL-6R beads were used as positive controls (direct binding). A low signal indicates the specificity of the candidates for IL-6R, while a high signal indicates no apparent affinity for AChBP or IgG. (B) Assessment of the candidates by biological assay. The candidate proteins were assessed for their potential to inhibit and/or enhance the proliferation of DS-1 cells (a human B-lymphoma cell line). IL-6R monoclonal antibody was used as a positive control for the inhibition of proliferation. Graphs are plotted as concentration of IL-6R mAb and peptides vs. % Growth. Open circles denote control (PBS in which IL-6R mAb and proteins were dissolved), filled diamonds denote IL-6R mAb (purple line), filled squares denote R10-13, inverted filled triangles denote R10-14, filled circles denote R10-15 (black line) and open triangles denote R10-17. (C) Assessment of the agonist activity of R10-14. The candidate protein, R10-14, which enhanced the proliferation of cells in (B), was assessed for its potential to enhance cell proliferation in the absence of IL-6; (i) shows the dose-dependent proliferation of cells by IL-6 and (ii) shows the dose-dependent proliferation of cells by R10-14. Proper controls such as PBS were included. ED50 for IL-6 and R10-14 were determined to be 15 ± 2 pM and 20 ± 6 nM, respectively.