A Novel Immunological Assay for Hepcidin Quantification in Human Serum

Abstract

BackgroundHepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera.Methods and FindingsAn ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L).ConclusionsWe describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.

Phagocytosis of gadolinium chloride or zymosan induces simultaneous upregulation of hepcidin- and downregulation of hemojuvelin- and Fpn-1-gene expression in murine liver

“…35 This conclusion, however, must be tempered by the fact that the ELISA measurement of pro-hepcidin may not detect a change in functional hepcidin. 38 The changes in gene expression of these iron metabolism-related proteins occurred in the absence of hepatotoxicity, as serum AST and ALT levels did not increase in these experiments.…”

Benefits and Risks of Iron Supplementation in Anemic Neonatal Pigs

“…Several suitable serum Hepc assays have been developed mostly for humans. 40,41,42 However, recent results of the first international round robin for the quantification of urinary and plasma hepcidin assays show that more work is needed for the use of Hepc plasma evaluation in experimental and clinical application. 43 Several cases of iron toxicity have been reported on the commonly practiced administration of large amounts of FeDex in a single intramuscular injection.…”

Hepcidin in Human Iron Disorders: Diagnostic Implications

“…In addition, Ganz et al (29 ) developed a competitive (c)ELISA for human serum hepcidin. Other groups have also reported reliable hepcidin assays that can be divided in 3 main methodologies: (a) MS (85)(86)(87)(88)(89)(90)(91)(92)(93)(94)(95); (b) immunochemical assays, comprising cRIA (96,97 ), cELISA (13,29,98,99 ), and a 2-site ELISA (100 ); and (c) a ligand-binding assay (43 ) ( Table 1). Of the currently available commercial immunochemical research kits for serum hepcidin, we found the RIA and enzyme-immunoassay kits of Bachem (purchased November 2009 and August 2010, respectively) to be suitable to differentiate between hepcidin concentrations in serum samples of controls and patients with various iron disorders, whereas the bioactive hepcidin kit of DRG Instruments, (purchased October 2009) gave similar concentrations for all samples and could not discriminate between iron metabolism disorders (unpublished results).…”