Antihepcidin antibody treatment neutralized hepcidin in vitro and in vivo. (A) By Western analysis, Ab2.7 detected purified shHepc and rhHepc, weakly detected smHepc1, and did not detect a defensin 1 control (Sigma-Aldrich). Ponceau S staining of blots was conducted to demonstrate protein loading. Some precipitation of smHepc1 occurred in reducing conditions, evident by decreased Ponceau S staining. (B) Hepcidin treatment (rhHepc) increased intracellular iron, measured as blue/green ratio with an iron-responsive β-lactamase reporter gene; EC₅₀ 40nM (n = 3 wells/point). (C) Ab2.7 neutralized hepcidin activity measured as in panel B (37nM rhHepc); IC₅₀ 14nM. (D) Ab2.7 prevented hepcidin-mediated serum iron decrease in mice. Subcutaneous Ab2.7 administered 3 days before 25 μg of hepcidin (IP); serum iron measured 2 hours later (n = 5/group). All statistical differences from rhHepc and control antibody group (□) are shown: *P < .05; ***P < .001.