Identification and synthesis of the peptide that binds hepcidin. (A) A schema to identify and synthesize a peptide that binds hepcidin. A high-affinity peptide binding to hepcidin was identified using phage display selection and chemically synthesized. The resultant peptide was conjugated with PEG and designated as HBP. (B) Measurement of affinity of HBP for hepcidin by a biolayer interferometry–based binding assay. Biotin-hepcidin-25–loaded sensors captured HBP at concentrations ranging from 20.0 to 15.0μM. Sensorgrams represent 3 HBP concentrations (20.0μM, 17.5μM, and 15.0μM). A sensorgram of a control sample with no HBP was subtracted. (C) Comparison of HBP affinity for hepcidin between hepcidin-25 and hepcidin-20. Biotin-HBP loaded sensors captured hepcidin-25, hepcidin-20, or control peptide at a concentration of 20.0μM.