A Novel Immunological Assay for Hepcidin Quantification in Human Serum

Contains fulltext : 81054.pdf (publisher's version ) (Open Access)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. METHODS AND FINDINGS: 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

Clinical Value of Plasma Soluble Urokinase-Type Plasminogen Activator Receptor Levels in Term Neonates with Infection or Sepsis: A Prospective Study

Background. suPAR, the soluble form of the urokinase-type plasminogen activator receptor, has been identified as a biomarker of infection in adults but its properties in neonatal infection are not known. Methods. Plasma suPAR levels were determined by ELISA in 47 term neonates with infection (19 bacterial and 28 viral) and in 18 healthy neonates as controls. Thirteen out of 47 infected neonates were septic. In all infected neonates, suPAR levels were repeated at 24 hours, 48 hours, 3–5 days, and 7–10 days following admission. Results. Plasma suPAR levels were significantly increased in infected neonates upon admission, whereas they were highest in septic neonates, in comparison with controls P<0.001 and correlated positively with serum CRP levels (P=0.001). At infection subsidence, suPAR concentrations decreased significantly in comparison with baseline (P<0.001) but remained higher than in controls (P=0.01). Receiver operating characteristic analysis resulted in significant areas under the curve for detecting either infected or septic neonates, but not for discriminating between bacterial and viral cause of infection. Conclusions. suPAR is a diagnostic biomarker of infection or sepsis in term neonates; however, it cannot discriminate bacterial from viral infections and also its utility for monitoring the response to treatment is questioned

Clinical Value of Plasma Soluble Urokinase-Type Plasminogen Activator Receptor Levels in Term Neonates with Infection or Sepsis: A Prospective Study

Background. suPAR, the soluble form of the urokinase-type plasminogen activator receptor, has been identified as a biomarker of infection in adults but its properties in neonatal infection are not known. Methods. Plasma suPAR levels were determined by ELISA in 47 term neonates with infection (19 bacterial and 28 viral) and in 18 healthy neonates as controls. Thirteen out of 47 infected neonates were septic. In all infected neonates, suPAR levels were repeated at 24 hours, 48 hours, 3-5 days, and 7-10 days following admission. Results. Plasma suPAR levels were significantly increased in infected neonates upon admission, whereas they were highest in septic neonates, in comparison with controls (P &lt; 0.001) and correlated positively with serumCRP levels (P = 0.001). At infection subsidence, suPAR concentrations decreased significantly in comparison with baseline (P &lt; 0.001) but remained higher than in controls (P = 0.01). Receiver operating characteristic analysis resulted in significant areas under the curve for detecting either infected or septic neonates, but not for discriminating between bacterial and viral cause of infection. Conclusions. suPAR is a diagnostic biomarker of infection or sepsis in term neonates; however, it cannot discriminate bacterial from viral infections and also its utility for monitoring the response to treatment is questioned

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. Methods and Findings: 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 mu g/L and a detection limit of 5.4 mu g/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 mu g/L) and 10 patients with iron deficiency anemia (15.7 mu g/L) and higher in 7 patients with Hodgkin lymphoma (116.7 mu g/L) compared to 32 age-matched healthy controls (42.7 mu g/L). Conclusions: We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility

Intra-assay variation.

<p>Intra-assay variation.</p

Inter-assay variation.

<p>Inter-assay variation.</p

Recovery of calibrator added to human serum samples.

a<p>(Observed concentration/Expected concentration)×100.</p

Correlation between serum hepcidin and ferritin in healthy controls.

<p>Hepcidin values measured by our ELISA assay correlate significantly with ferritin levels. Pearson correlation: 0.474 (p = 0.006).</p

Hepcidin serum concentration in healthy controls (control), patients with iron deficiency anemia (IDA), juvenile haemochromatosis (JH) and Hodgkin's lymphoma (HL).

<p>Box plots show the 25th and 75th percentile with median value for each group. Minimum and maximum values are also depicted. Significant difference compared to control is indicated by asterisk (**p<0.010).</p

Dilution linearity of ELISA.

a<p>(Observed concentration/Expected concentration)×100.</p