A Dual-Monoclonal Sandwich ELISA Specific for Hepcidin-25

BACKGROUND: Hepcidin, a key regulator of iron metabolism, binds to the iron transporter ferroportin to cause its degradation. In humans, hepcidin deficiency has been linked to hemochromatosis and iron overload, whereas increased concentrations have been reported in anemia of cancer and chronic disease. There is currently an unmet clinical need for a specific immunoassay with a low limit of quantification to measure serum concentrations of hepcidin-25, the active form of the protein. METHODS: We generated 2 antihepcidin-25 monoclonal antibodies and used them to build a sandwich ELISA. We correlated ELISA results to hepcidin-25 measurements by LC-MS and used ELISA to measure serum hepcidin-25 concentrations in normal individuals, cancer patients, and patients with rheumatoid arthritis. RESULTS: The sandwich ELISA was highly specific for hepcidin-25, having a limit of quantification of 0.01 μg/L (10 pg/mL). Serum concentrations of hepcidin-25 measured by ELISA correlated with hepcidin-25 concentrations measured by using an independent LC-MS assay ( r = 0.98, P < 0.001). Hepcidin-25 concentrations were increased in patients with cancer (median 54.8 μg/L, 25%–75% range 23.2–93.5 μg/L, n = 34) and rheumatoid arthritis (median 10.6 μg/L, 25%–75% range 5.9–18.4 μg/L, n = 76) compared with healthy individuals (median 1.20 μg/L, 25%–75% range 0.42–3.07 μg/L, n = 100). CONCLUSIONS: The use of 2 monoclonal antibodies in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of the active form of hepcidin. This ELISA should help to improve our understanding of the role of hepcidin in regulating iron metabolism.