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. 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.

Altered angiogenesis in preeclampsia: evaluation of a new test system for measuring placental growth factor.

BACKGROUND: Decreased concentrations of the circulating angiogenic factors, free placental growth factor (PLGF) and free vascular endothelial growth factor (VEGF), and increased concentrations of the anti-angiogenic factor, soluble fms-like tyrosine kinase 1 (sFLT-1) have been observed during clinical preeclampsia. We established a new PLGF-ELISA kit for the measurement of PLGF in sera. In the present study, we demonstrated the assay characteristics by measurement of PLGF expression in normal and preeclamptic pregnancies as compared to an established research kit. METHODS: Blood samples were taken from 64 women with singleton uncomplicated pregnancies for longitudinal measurement of PLGF in the course of pregnancy. In 30 preeclamptic patients, serum levels of PLGF and sFLT-1 were measured by Human PLGF-ELISA and Human sVEGF R1 ELISA according to the described test principles. The assay characteristics of the new PLGF-ELISA were determined and the results were compared to those performed with an available research kit. RESULTS: The PLGF concentration in normal pregnancies showed a steady increase starting at the beginning of the second trimester with a peak at 28-32 weeks and a consistent decline thereafter. The preeclamptic pregnancies had significant lower serum concentrations of PLGF and significant higher serum concentrations of sFLT-1 as compared to the non-preeclamptic pregnancies. All the measured assay characteristics fulfilled the required specifications. Comparison of the values of the new PLGF-ELISA and the established research kit resulted in a correlation coefficient of 0.921. CONCLUSIONS: Our results support the hypothesis that an imbalance between factors promoting angiogenesis, such as PLGF, and factors antagonizing angiogenesis, such as sFLT-1, has a fundamental role in the pathogenesis of preeclampsia. The new established ELISA test can be considered reliable and it offers many advantages. As it is authorized for routine diagnostic testing, it may offer new possibilities in the prediction of preeclampsia in clinical routine.