Competitive Displacement Of Phage Clones Expressing hTNFR2-Hotspot Peptide Ligands By Human TNF (Alpha And Beta) Or Soluble TNF Receptor (R1 Or R2) Fusions. Recombinant human TNFR2 was coated on MaxiSorp 96 well plates and blocked with PBS containing 2% non-fat milk as previously described (3). Phage clones expressing Hotspot Peptide Ligands (phage clones at their ED50 values) are added in the presence of varying concentrations of competitor (e.g., TNF or soluble TNF receptor). Panel A) KcC7 phage competition, Panel B) KcD11 phage, Panel C) KcF6 phage, Panel D) KcF12 phage. IC50 values are determined by probing for phage binding (using HRP labeled anti-M13 antibody) to TNFR2 in the presence of competitor. Data is presented as percent inhibition and to determine binding kinetic parameters data were fit to a four-parameter logistic equation using Xlfit™, an add-in for Microsoft Excel.