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

  • Original Article—Liver, Pancreas, and Biliary Tract
  • Published:
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Abstract

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.

Conclusion

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.

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References

  1. Krause A, Neitz S, Mägert HJ, Schulz A, Forssmann WG, Schulz-Knappe P, et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett. 2000;480:147–50.

    Article  PubMed  CAS  Google Scholar 

  2. Park CH, Valore EV, Waring AJ, Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem. 2001;276:7806–10.

    Article  PubMed  CAS  Google Scholar 

  3. Kulaksiz H, Gehrke SG, Janetzko A, Rost D, Bruckner T, Kallinowski B, et al. Prohepcidin: expression and cell specific localisation in the liver and its regulation in hereditary haemochromatosis, chronic renal insufficiency, and renal anaemia. Gut. 2004;53:735–43.

    Article  PubMed  CAS  Google Scholar 

  4. Valore EV, Ganz T. Posttranslational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin. Blood Cells Mol Dis. 2008;40:132–8.

    Article  PubMed  CAS  Google Scholar 

  5. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306:2090–3.

    Article  PubMed  CAS  Google Scholar 

  6. Anderson GJ, Frazer DM, McLaren GD. Iron absorption and metabolism. Curr Opin Gastroenterol. 2009;25:129–35.

    Article  PubMed  CAS  Google Scholar 

  7. Pietrangelo A. Hemochromatosis: an endocrine liver disease. Hepatology. 2007;46:1291–301.

    Article  PubMed  CAS  Google Scholar 

  8. Figura G, Kulaksiz H. Diagnosis and treatment in hereditary hemochromatosis. Dtsch Med Wochenschr. 2009;134:2565–72.

    Article  Google Scholar 

  9. Fujita N, Sugimoto R, Takeo M, Urawa N, Mifuji R, Tanaka H, et al. Hepcidin expression in the liver: relatively low level in patients with chronic hepatitis C. Mol Med. 2007;13:97–104.

    Article  PubMed  CAS  Google Scholar 

  10. van der Putten K, Braam B, Jie KE, Gaillard CA. Mechanism of disease: erythropoietin resistance in patients with both heart and kidney failure. Nat Clin Pract Nephrol. 2008;4:47–57.

    Article  PubMed  Google Scholar 

  11. Ashby DR, Gale DP, Busbridge M, Murphy KG, Duncan ND, Cairns TD, et al. Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal disease. Kidney Int. 2009;75:976–81.

    Article  PubMed  CAS  Google Scholar 

  12. Weiss G, Theurl I, Eder S, Koppelstaetter C, Kurz K, Sonnweber T, et al. Serum hepcidin concentration in chronic haemodialysis patients: associations and effects of dialysis, iron and erythropoietin therapy. Eur J Clin Invest. 2009;10:883–90.

    Article  Google Scholar 

  13. Young MF, Glahn RP, Ariza-Nieto M, Inglis J, Olbina G, Westerman M, et al. Serum hepcidin is significantly associated with iron absorption from food and supplemental sources in healthy young women. Am J Clin Nutr. 2009;89:533–8.

    Article  PubMed  CAS  Google Scholar 

  14. Zoller H, McFarlane I, Theurl I, Stadlmann S, Nemeth E, Oxley D, et al. Primary iron overload with inappropriate hepcidin expression in V162del ferroportin disease. Hepatology. 2005;42:466–72.

    Article  PubMed  CAS  Google Scholar 

  15. Kulaksiz H, Theilig F, Bachmann S, Gehrke SG, Rost D, Janetzko A, et al. The iron-regulatory peptide hormone hepcidin: expression and cellular localization in the mammalian kidney. J Endocrinol. 2005;184:361–70.

    Article  PubMed  CAS  Google Scholar 

  16. Tomosugi N, Kawabata H, Wakatabe R, Higuchi M, Yamaya H, Umehara H, et al. Detection of serum hepcidin in renal failure and inflammation by using ProteinChip System. Blood. 2006;108:1381–7.

    Article  PubMed  CAS  Google Scholar 

  17. Kemna EH, Tjalsma H, Podust VN, Swinkels DW. Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications. Clin Chem. 2008;53:620–8.

    Article  Google Scholar 

  18. Kobold U, Dülffer T, Dangl M, Escherich A, Kubbies M, Röddiger R, et al. Quantification of hepcidin-25 in human serum by isotope dilution micro-HPLC-tandem mass spectrometry. Clin Chem. 2008;54:1584–6.

    Article  PubMed  CAS  Google Scholar 

  19. Ward DG, Roberts K, Stonelake P, Goon P, Zampronio CG, Martin A, et al. SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labeled hepcidin as an internal standard. Proteome Sci. 2008;14:28.

    Article  Google Scholar 

  20. Li H, Rose MJ, Tran L, Zhang J, Miranda LP, James CA, et al. Development of a method for the sensitive and quantitative determination of hepcidin in human serum using LC-MS/MS. J Pharmacol Toxicol Methods. 2009;59:171–80.

    Article  PubMed  CAS  Google Scholar 

  21. Castagna A, Campostrini N, Zaninotto F, Girelli D. Hepcidin assay in serum by SELDI-TOF-MS and other approaches. J Proteomics. 2010;73:527–36.

    Article  PubMed  CAS  Google Scholar 

  22. Grebenchtchikov N, Geurts-Moespot AJ, Kroot JJ, den Heijer M, Tjalsma H, Swinkels DW, et al. High-sensitive radioimmunoassay for human serum hepcidin. Br J Haematol. 2009;146:317–25.

    Article  PubMed  CAS  Google Scholar 

  23. Busbridge M, Griffith C, Ashby D, Gale D, Jayantha A, Sanwaiya A, et al. Development of a novel immunoassay for the iron regulatory peptide hepcidin. Br J Biomed Sci. 2009;66:150–7.

    PubMed  CAS  Google Scholar 

  24. Koliaraki V, Marinou M, Vassilakopoulos TP, Vavourakis E, Tsochatzis E, Pangalis GA, et al. A novel immunological assay for hepcidin quantification in human serum. PLoS ONE. 2009;4:e4581. doi:10.1371/journal.pone.0004581.

    Article  PubMed  Google Scholar 

  25. Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M. Immunoassay for human serum hepcidin. Blood. 2008;112:4292–7.

    Article  PubMed  CAS  Google Scholar 

  26. Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot P, et al. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem. 2001;276:7811–9.

    Article  PubMed  CAS  Google Scholar 

  27. Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256:495–7.

    Article  PubMed  Google Scholar 

  28. Schwarz P, Strnad P, Singer N, Oswald F, Ehehalt R, Adler G, et al. Identification, sequencing, and cellular localization of hepcidin in guinea pig (Cavia porcellus). J Endocrinol. 2009;202:389–96.

    Article  PubMed  CAS  Google Scholar 

  29. Kroot JJ, Kemna EH, Bansal SS, Busbridge M, Campostrini N, Girelli D, et al. Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization. Haematologica. 2009;94:1748–52.

    Article  PubMed  CAS  Google Scholar 

  30. Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, et al. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest. 2002;110:1037–44.

    PubMed  CAS  Google Scholar 

  31. Nicolas G, Viatte L, Bennoun M, Beaumont C, Kahn A, Vaulont S. Hepcidin, a new iron regulatory peptide. Blood Cells Mol Dis. 2002;29:327–35.

    Article  PubMed  CAS  Google Scholar 

  32. Lin L, Valore EV, Nemeth E, Goodnough JB, Gabayan V, Ganz T. Iron transferrin regulated hepcidin synthesis in primary hepatocytes culture through hemojuvelin and BMP2/4. Blood. 2007;110:2182–9.

    Article  PubMed  CAS  Google Scholar 

  33. Kroot JJ, Hendriks JC, Laarakkers CM, Klaver SM, Kemna EH, Tjalsma H, et al. (Pre)analytical imprecision, between-subject variability, and daily variations in serum and urine hepcidin: implications for clinical studies. Anal Biochem. 2009;389:124–9.

    Article  PubMed  CAS  Google Scholar 

  34. Dale JC, Burritt MF, Zinsmeister AR. Diurnal variation of serum iron, iron-binding capacity, transferrin saturation, and ferritin levels. Am J Clin Pathol. 2002;117:802–8.

    Article  PubMed  CAS  Google Scholar 

  35. Fujita N, Sugimoto R, Urawa N, Araki J, Mifuji R, Yamamoto M, et al. Hepatic iron accumulation is associated with disease progression and resistance to interferon/ribavirin combination therapy in chronic hepatitis C. J Gastroenterol Hepatol. 2007;22:1886–93.

    Article  PubMed  CAS  Google Scholar 

  36. Franchini M, Targher G, Capra F, Montagnana M, Lippi G. The effect of iron depletion on chronic hepatitis C virus infection. Hepatol Int. 2008;2:335–40.

    Article  PubMed  Google Scholar 

  37. Trinder D, Ayonrinde OT, Olynyk JK. HCV, iron, and oxidative stress: the new choreography of hepcidin. Gastroenterology. 2008;134:348–51.

    Article  PubMed  CAS  Google Scholar 

  38. Fujita N, Sugimoto R, Motonishi S, Tomosugi N, Tanaka H, Takeo M, et al. Patients with chronic hepatitis C achieving a sustained virological response to peginterferon and ribavirin therapy recover from impaired hepcidin secretion. J Hepatol. 2008;49:702–10.

    Article  PubMed  CAS  Google Scholar 

  39. Girelli D, Pasino M, Goodnough JB, Nemeth E, Guido M, Castagna A, et al. Reduced serum hepcidin levels in patients with chronic hepatitis C. J Hepatol. 2009;51:845–52.

    Article  PubMed  CAS  Google Scholar 

  40. Miura K, Taura K, Kodama Y, Schnabl B, Brenner DA. Hepatitis C virus-induced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity. Hepatology. 2008;48:1420–9.

    Article  PubMed  CAS  Google Scholar 

  41. Nishina S, Hino K, Korenaga M, Vecchi C, Pietrangelo A, Mizukami Y, et al. Hepatitis C virus-induced reactive oxygen species raise hepatic iron level in mice by reducing hepcidin transcription. Gastroenterology. 2008;134:226–38.

    Article  PubMed  CAS  Google Scholar 

  42. Gehrke SG, Kulaksiz H, Hermann T, Rieder HD, Bents K, Veltkamp C, et al. Expression of hepcidin in hereditary hemochromatosis: evidence for a regulation in response to the serum transferrin saturation and to non-transferrin-bound iron. Blood. 2003;102:371–6.

    Article  PubMed  CAS  Google Scholar 

  43. Piperno A, Girelli D, Nemeth E, Trombini P, Bozzini C, Poggiali E, et al. Blunted hepcidin response to oral iron challenge in HFE-related hemochromatosis. Blood. 2007;110:4096–100.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Maria C. D. Rasenack and Rayan I. Habib for performing Western blot analyses and critically reading the manuscript. This work was supported by Deutsche Forschungsgemeinschaft grants KU 1253/5-3 (H.K. and P.S.) and STR 1095/2-1 (P.St.).

Conflict of interest

A.J. is an employee and H.K. serves as a scientific advisor of DRG Instruments GmbH, which is engaged in the commercial development of the assay described in this manuscript.

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Authors and Affiliations

  1. Division of Gastroenterology, Department of Internal Medicine, University Hospital Ulm, Albert-Einstein-Allee 23, 89081, Ulm, Germany

    Peggy Schwarz, Pavel Strnad, Guido von Figura, Guido Adler & Hasan Kulaksiz

  2. DRG Instruments GmbH, Marburg, Germany

    Alfred Janetzko

  3. Department of Internal Medicine, University Hospital, Zurich, Switzerland

    Pierre Krayenbühl

Authors
  1. Peggy Schwarz
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  2. Pavel Strnad
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  7. Hasan Kulaksiz
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Correspondence to Hasan Kulaksiz.

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Schwarz, P., Strnad, P., von Figura, G. et al. A novel monoclonal antibody immunoassay for the detection of human serum hepcidin. J Gastroenterol 46, 648–656 (2011). https://doi.org/10.1007/s00535-010-0344-z

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  • DOI: https://doi.org/10.1007/s00535-010-0344-z

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