Characterization of hepcidin-binding Abs and development of a sandwich ELISA. (A) Polyclonal Abs against human hepcidin demonstrated that construction of a sandwich ELISA was possible. Each polyclonal Ab (4364 and 4366) was tested as both the capture Ab (eg, 4364 bound to ELISA plate) and detection Ab (4364 conjugated to horseradish peroxidase for detection). (B) The ability of 2 Abs to bind to hepcidin simultaneously was profiled by Biacore. Representative plot showing a panel of Abs, some of which bound to Ab 19D12 coated on a chip and treated with hepcidin. Binding of the second Ab was demonstrated by an increase in detected mass (increase in relative units [RU] of binding). (C) Diagram categorizing hepcidin-binding Abs into classes based on ability to recognize overlapping or distinct epitopes on hepcidin. Solid line indicates classes that could bind hepcidin simultaneously. (D) Comparison of performance of anti-human hepcidin sandwich ELISA with hepcidin detection by LC-MS/MS. Serum hepcidin concentrations from CKD patient samples were measured in both assays and compared (n = 46). Hepcidin ELISA (x-axis) and LC-MS/MS (y-axis).