Hepcidin 25 human enzyme immunoassay kit

Plasma levels of soluble transferrin receptor (sTFR) and ferritin were measured by enzyme immunoassay (Ramco Laboratories Inc, Houston, TX); plasma alpha-1-acid glycoprotein (AGP) and C-reactive protein (CRP) were measured by ELISA (R&D Systems Inc, Minneapolis, MN).
Hepcidin was measured in plasma by competition ELISA, using the hepcidin-25 (human) enzyme immunoassay kit (S-1337; Bachem, San Carlos, CA) with detection range 0.02–25 ng/mL, according to the manufacturer’s protocol. Plasma was diluted 1 in 4 in peptide-cleared human serum. Standards were run in duplicate and samples in singlicate, according to the manufacturer’s protocol. Samples giving readings outside the linear region of the curve were re-run at alternative dilutions. The intra-assay CV was mean 6.3% (range 5.7–6.9%), and inter-assay CV was 6.3%.
Iron deficiency was defined according to WHO guidelines, using a combination of low ferritin (< 12 μg/L in the absence of inflammation (CRP ≤ 5 mg/L) or < 30 μg/L in the presence of inflammation (CRP > 5 mg/L)) [30 , 31 ] plus evidence of iron depletion in the tissues, as evidenced by a sTfR/log₁₀ ferritin (sTfR-F) index >2 [18 (link)]. Anemia of inflammation (AI) was defined as hemoglobin < 105 g/L at 3 and 6 months or < 100 g/L at 12 months and CRP >5 mg/L, with no iron deficiency (ferritin > 30 μg/L and sTfR/log₁₀ ferritin (sTfR-F) index < 2).

Hepcidin concentrations in patient's plasma/serum samples were quantified by competition enzyme-linked immunoassay (C-ELISA) using the hepcidin-25 (human) enzyme immunoassay kit (Bachem, Torrance, CA, USA) according to the manufacturer's protocol. Samples and standards were run in duplicate. Plasma samples were diluted 1 in 10 or 1 in 50 in supplied standard diluent (peptide-cleared human serum) and analyzed using a 10-point twofold serial dilution (maximum concentration, 25 ng/mL) standard curve. Hepcidin concentrations were interpolated from standard curves generated by a four-parameter logistic nonlinear regression model using Prism (GraphPad Software Inc., La Jolla, CA). Appropriate dilutions were used to obtain readings inside the linear region of the curve.

Hepcidin-25 levels were measured using Hepcidin-25 (human) – Enzyme Immunoassay kit (Bachem, UK) with the high-sensitivity protocol and following manufacturer's instructions. Briefly, samples were diluted 1/5 in standard diluent, and added to an immunoplate pre-coated with anti-hepcidin together with rabbit anti-serum. Plates were incubated for 2 hours at room temperature then washed 5 times with the manufacturer's wash buffer. One hour incubation with streptavidin-HRP was followed by a wash step and further hour's incubation with TMB solution. Reactions were then terminated using hydrochloric acid (2N) and the optical density (OD) read at 450 nm. Human Hepcidin-25 peptide was used as a standard and analysis was carried out according to the manufacturer's instructions.