Human Hepcidin Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human Hepcidin Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human Hepcidin in cell culture supernates, serum, plasma, and urine. It contains synthetic Hepcidin and antibodies raised against synthetic Hepcidin. This immunoassay has been shown to accurately quantitate synthetic and naturally occurring human Hepcidin.

Preparation and Storage

Background: Hepcidin

Hepcidin, also known as Liver Expressed Antimicrobial Protein 1 (LEAP-1), is a peptide hormone that is involved in the regulation of iron metabolism (1, 2). It is synthesized as a preprohormone that is cleaved intracellularly and secreted as a mature 25 amino acid peptide (1, 3, 4). Hepcidin contains eight cysteine residues that form four disulfide bonds which appear to be important for stability in biological fluids (5). It is predominantly expressed, processed, and secreted by hepatocytes (2, 6). Hepcidin expression is positively regulated by inflammation via IL-6/JAK2/ STAT3 signaling, endoplasmic reticulum stress, and BMP-6 (7-11). BMP-6-dependent Hepcidin induction involves RGM-C/Hemojuvelin, which acts as a co-receptor for BMP-6 (11-13). Conversely, Hepcidin expression is negatively regulated by MMP-15/MT2-MMP and multiple erythropoietic stimuli, including anemia, hypoxia, and Erythropoietin (14-18). MMP-15 downregulates Hepcidin expression by interacting with and cleaving RGM-C (19).

Hepcidin was originally identified in human blood and urine as an antimicrobial peptide (1, 3). It has since been shown to regulate iron metabolism. Hepcidin binds the cellular iron exporter Ferroportin, and this interaction results in Ubiquitin-mediated degradation of both Hepcidin and Ferroportin (20-22). Degradation of Ferroportin results in reduced iron release from macrophages, hepatocytes, and duodenal enterocytes, suggesting that Hepcidin may be an effector of inflammatory hypoferremia (20). Pathologies have been associated with both deficiency and overexpression of Hepcidin. Overexpression of Hepcidin results in lower iron levels and may contribute to the development of anemia in many human disorders (23). Higher Hepcidin levels have also been linked to poor survival in patients with non-Hodgkin lymphoma (24). Hepcidin deficiency results in increased iron levels and is the pathogenic cause of iron overload in most forms of hereditary hemochromatosis (25, 26).

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.