Immunoassay for Human Hepcidin in Blood

Abstract

We developed and validated the first ELISA for hepcidin, the principal iron-regulatory hormone that has been very difficult to measure. The competitive ELISA uses a biotin-tagged hepcidin as a tracer to quantify hepcidin in serum, plasma or urine. In healthy volunteers, the 5–95% range of serum hepcidin concentrations was 29–254 ng/ml in men (n=65) and 17–286 ng/ml in women (n=49), with median concentrations 112 vs. 65, p<0.001. The lower limit of detection was 5 ng/ml. Serum, EDTA plasma and heparin plasma hepcidin measurements were in agreement (r> 0.98). Serum and urinary hepcidin concentrations in 24 healthy subjects correlated well (r=0.82). Serum hepcidin appropriately correlated with serum ferritin (r=0.63) reflecting the regulation of both proteins by iron stores. Healthy volunteers showed a diurnal increase of serum hepcidin at noon and 8pm compared to 8am, and a transient rise of serum hepcidin in response to iron ingestion. In 18 young healthy women, serum hepcidin correlated well (p<0.05) with intestinal iron absorption both from a food (r=0.49) and supplemental iron source (r=0.51), as determined by a stable iron isotope technique. In a variety of clinical conditions associated with iron disturbances we observed the appropriate changes in hepcidin concentrations: undetectable or low in patients with iron deficiency anemia (ferritin<10 ng/ml), treated HFE hemochromatosis, and juvenile hemochromatosis, and abnormally high in patients with inflammation (CRP>10 mg/dl), multiple myeloma, or chronic kidney disease. The new blood hepcidin ELISA yields accurate and reproducible measurements that appropriately reflect physiologic, pathologic and genetic influences, and is informative about the etiology of iron disorders.