A novel immunological assay for hepcidin quantification in human serum.
BACKGROUND
Hepcidin 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.


RESULTS
An 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 microg/L and a detection limit of 5.4 microg/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 microg/L) and 10 patients with iron deficiency anemia (15.7 microg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 microg/L) compared to 32 age-matched healthy controls (42.7 microg/L).


CONCLUSIONS
We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.
Koliaraki
Vasiliki
V
Department of Biochemistry, Laboratory of Molecular Biology and Immunobiotechnology, Hellenic Pasteur Institute, Athens, Greece.
Marinou
Martha
M
Vassilakopoulos
Theodoros P
TP
Vavourakis
Eustathios
E
Tsochatzis
Emmanuel
E
Pangalis
Gerassimos A
GA
Papatheodoridis
George
G
Stamoulakatou
Alexandra
A
Swinkels
Dorine W
DW
Papanikolaou
George
G
Mamalaki
Avgi
A
D000740:Anemia / Q000175:diagnosis*; D018798:Anemia, Iron-Deficiency / Q000097:blood; D023181:Antimicrobial Cationic Peptides / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006432:Hemochromatosis / Q000097:blood; D064451:Hepcidins; D006689:Hodgkin Disease / Q000097:blood; D006801:Humans; D012680:Sensitivity and Specificity
D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins