A novel monoclonal antibody immunoassay for the detection of human serum hepcidin

Autor: Alfred Janetzko, Guido Adler, Pierre Krayenbühl, Hasan Kulaksiz, Peggy Schwarz, Pavel Strnad, Guido von Figura
Přispěvatelé: University of Zurich, Kulaksiz, H
Jazyk: angličtina
Rok vydání: 2011
Předmět:
Adult
Male
medicine.drug_class
Blotting
Western
Immunoblotting
10265 Clinic for Endocrinology and Diabetology
Dot blot
Fluorescent Antibody Technique
Enzyme-Linked Immunosorbent Assay
610 Medicine & health
Monoclonal antibody
Sensitivity and Specificity
Mice
Young Adult
Western blot
Hepcidins
Hepcidin
hemic and lymphatic diseases
Medicine
Animals
Humans
2715 Gastroenterology
Aged
Mice
Inbred BALB C
biology
medicine.diagnostic_test
business.industry
Transferrin saturation
Gastroenterology
nutritional and metabolic diseases
Antibodies
Monoclonal
Reproducibility of Results
Middle Aged
Molecular biology
Hepatitis C
Hereditary hemochromatosis
Immunoassay
Case-Control Studies
biology.protein
Female
Hemochromatosis
Antibody
10029 Clinic and Policlinic for Internal Medicine
business
Antimicrobial Cationic Peptides
Popis: 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. 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). 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. 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.
Databáze: OpenAIRE
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