Validation of a competitive ELISA assay for the quantification of human serum hepcidin.
BACKGROUND
Hepcidin-25 is a potential marker for iron disorders with a demand for accessible assays. This study aimed to evaluate a commercial competitive enzyme-linked immunosorbent assay (cELISA) for hepcidin quantitation.


METHODS
Serum samples; 95 healthy subjects (HS), six patients with iron deficiency (ID), 84 patients with liver disorders (LD) and 220 hemodialysis patients (HD), were analyzed. Controls were used for imprecision, while accuracy was evaluated by quantitating hepcidin-25 with LC-MS/MS in 149 samples. Cross-reactivity for hepcidin-20 and hepcidin-22 was tested. Hepcidin-mRNA expression in 37 liver biopsies was measured.


RESULTS
S-hepcidin ranged from 8-76 and 2-31 μg/L in healthy men and women. Levels in ID, LD and HD significantly differed from HS. Total coefficients of variation (CV) for controls were 24% and 22%. Within-sample CV was 10%. Despite a good correlation with LC-MS/MS (r = 0.89), the cELISA showed higher values and detected hepcidin-20 and hepcidin-22. Hepcidin-mRNA correlated well with S-hepcidin using cELISA and LC-MS/MS (r = 0.69 and 0.64).


CONCLUSIONS
The correlation with LC-MS/MS is good and the examined kit can differentiate between patient groups although it is not specific for hepcidin-25. Considering ELISA's capacity to readily be set up, the investigated kit can be applied. Specific reference ranges are required.
a Department of Clinical Chemistry , Karolinska University Hospital & Institution for Laboratory Medicine , Stockholm , Sweden.
Gunilla
G
Dahlfors
b Division of Gastroenterology and Hepatology , Karolinska University Hospital & Department of Medicine , Stockholm , Sweden.
Per
P
Stål
a Department of Clinical Chemistry , Karolinska University Hospital & Institution for Laboratory Medicine , Stockholm , Sweden.
Eva C
EC
Hansson
c Renal Medicine, CLINTEC, Karolinska Institute , Stockholm , Sweden.
Peter
P
Bàràny
a Department of Clinical Chemistry , Karolinska University Hospital & Institution for Laboratory Medicine , Stockholm , Sweden.
Christin
C
Sisowath
a Department of Clinical Chemistry , Karolinska University Hospital & Institution for Laboratory Medicine , Stockholm , Sweden.
Liselotte
L
Onelöv
d Department of Clinical Chemistry , Helsingborg, University and Regional Laboratories Region Skåne , Stockholm , Sweden.
Dick
D
Nelson
a Department of Clinical Chemistry , Karolinska University Hospital & Institution for Laboratory Medicine , Stockholm , Sweden.
Gösta
G
Eggertsen
e Department of Gastroenterology , Ersta Hospital , Stockholm , Sweden.
Joel
J
Marmur
a Department of Clinical Chemistry , Karolinska University Hospital & Institution for Laboratory Medicine , Stockholm , Sweden.
Soheir
S
Beshara
D000328:Adult; D000368:Aged; D001774:Blood Chemical Analysis; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D064451:Hepcidins / Q000097:blood* / Q000235:genetics; D006801:Humans; D007676:Kidney Failure, Chronic / Q000097:blood; D057230:Limit of Detection; D008099:Liver / Q000378:metabolism; D008107:Liver Diseases / Q000097:blood; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D055815:Young Adult
C578702:HAMP protein, human; D064451:Hepcidins; D012333:RNA, Messenger
LC-MS/MS; cELISA; hepcidin-20; hepcidin-22; hepcidin-mRNA