A novel monoclonal antibody immunoassay for the detection of human serum hepcidin.
BACKGROUND
Hepcidin is a liver-derived peptide hormone regulating iron metabolism. Changes in the expression of hepcidin are known to be the key pathogenic factors in hereditary hemochromatosis and are associated with infection and inflammation. To better understand the hormone's function in human disease, we aimed to establish an immunoassay to determine hepcidin concentrations in serum.


METHODS
Monoclonal antibodies mHK(8) and mHK(9) were generated and characterized by dot blot, Western blot, and immunofluorescence. A competitive enzyme-linked immunosorbent assay (ELISA) was established with mHK(9).


RESULTS
Both antibodies recognized hepcidin, by dot blot and Western blot, respectively. In human liver, mHK(8)/(9) showed an immunofluorescence staining pattern in hepatocytes identical to that of established prohepcidin antibodies. The developed immunoassay with mHK(9), reliably detecting mature hepcidin in serum over a large concentration range (0.9-140 ng ml⁻¹), showed high sensitivity and precision (intra-/interassay coefficients of variation: 4-5 and 7-11%; mean linearity: 85-112%; mean recovery: 87-114%). To test the clinical functionality of the developed assay we measured hepcidin serum concentrations in healthy volunteers, hepatitis C virus (HCV) patients, and two groups of hemochromatotic patients undergoing phlebotomy. The assay distinguished low hepcidin level in HCV and homozygous hemochromatosis patients from normal-range controls and compound heterozygous hemochromatosis patients. In healthy subjects and HCV patients, hepcidin levels were correlated with iron and transferrin saturation; no correlation was observed in the hemochromatotic patients.


CONCLUSIONS
We developed a monoclonal antibody ELISA that quantifies serum hepcidin levels with high sensitivity, robustness, and reliability of detection. The hepcidin ELISA should help to enhance our understanding of hepcidin-related human disorders.
Division of Gastroenterology, Department of Internal Medicine, University Hospital Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany.
Peggy
P
Schwarz
Pavel
P
Strnad
Guido
G
von Figura
Alfred
A
Janetzko
Pierre
P
Krayenbühl
Guido
G
Adler
Hasan
H
Kulaksiz
D000328:Adult; D000368:Aged; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D023181:Antimicrobial Cationic Peptides / Q000097:blood* / Q000276:immunology; D015153:Blotting, Western; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D006432:Hemochromatosis / Q000097:blood*; D006526:Hepatitis C / Q000097:blood; D064451:Hepcidins; D006801:Humans; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055815:Young Adult
D000911:Antibodies, Monoclonal; D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; C579599:Hamp protein, mouse; D064451:Hepcidins