All children aged 2–6 years old were recruited from ten rural villages in the West Kiang region of The Gambia during the malaria season (July to August 2001)42 (link). We used cross-sectional data collected at the start of the malaria season. All children had a clinical examination, anthropometric measurements and a 3-d course of mebendazole for possible hookworm infection. A blood sample was collected for complete blood count, observations with a malaria slide, measurements of ferritin (Microparticle Enzyme Immunoassay (Abbott Architect)), hepcidin (Hepcidin-25 (human) EIA Kit (Bachem) and ACT (immunoturbidimetry, Cobas Mira Plus Bio-analyzer, Roche) levels and DNA extraction. Children with a temperature >37.5°C had a malaria blood film, appropriate clinical treatment and a blood sample 2 weeks later after recovery from illness. Genotyping of sickle cell was performed on amplified DNA as detailed elsewhere42 (link). The Gambian Bachem hepcidin values were harmonized by converting to the old DRG hepcidin assay values ((0.266×Bachem values)+1.633) and then to the new High Sensitive DRG hepcidin assay values ((1.989× old DRG values)-3.24) as previously validated43 (link).
Hepcidin 25 human eia kit
Manufactured by Bachem
Hepcidin-25 (human) EIA Kit is a quantitative in vitro diagnostic enzyme-linked immunosorbent assay (EIA) for the measurement of hepcidin-25 concentration in human serum or plasma samples. The kit includes a 96-well microplate pre-coated with an antibody specific to human hepcidin-25, as well as the necessary reagents and solutions to perform the assay.
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5 protocols using hepcidin 25 human eia kit
1
Cross-Sectional Study of Malaria, Anemia, and Hepcidin in Gambian Children
2
Measuring Malaria Biomarkers in Humans
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Plasmodium falciparum parasitaemia was determined as previously described (Nyakeriga et al., 2004 (link)). Haemoglobin typing (HbA and HbS) was by electrophoresis (Helena Laboratories, Beaumont, TX) while α-thalassemia genotyping was by PCR (Chong et al., 2000 (link)). Plasma concentrations of ferritin, soluble transferrin receptor (sTfR) and C-reactive protein (CRP) were determined as previously described (Atkinson et al., 2014 (link), Nyakeriga et al., 2004 (link)). IgG antibodies against whole P. falciparum schizont extract and against the 3D7 allele of apical membrane antigen 1 (AMA1) and merozoite surface protein 2 (MSP2) were assayed by enzyme linked immunosorbent assay (ELISA) (Mugyenyi et al., 2013 (link)).
Plasma hepcidin was quantified by competitive ELISA (Hepcidin-25 (human) EIA Kit, Bachem) (Atkinson et al., 2014 (link)). Standards and samples were analyzed in duplicate or triplicate. Samples giving readings outside the standard linear region were repeated at appropriate dilutions. Readings with coefficient of variation > 10% were repeated. The lower limit of detection (LOD) of hepcidin was estimated at 0.08 ng/ml based on the hepcidin value corresponding to 3 standard deviations below the mean no hepcidin blank optical density at 450 nm; undiluted samples giving reading of < LOD were reported as LOD/2 = 0.04 ng/ml.
Plasma hepcidin was quantified by competitive ELISA (Hepcidin-25 (human) EIA Kit, Bachem) (Atkinson et al., 2014 (link)). Standards and samples were analyzed in duplicate or triplicate. Samples giving readings outside the standard linear region were repeated at appropriate dilutions. Readings with coefficient of variation > 10% were repeated. The lower limit of detection (LOD) of hepcidin was estimated at 0.08 ng/ml based on the hepcidin value corresponding to 3 standard deviations below the mean no hepcidin blank optical density at 450 nm; undiluted samples giving reading of < LOD were reported as LOD/2 = 0.04 ng/ml.
3
Comprehensive Hematological Biomarker Profiling
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Haemoglobin (Medonic CA 530 Haemoglobinometer) and zinc protoporphyrin (ZnPP) levels (Aviv Biomedical Hematoflurometer) were measured within 24 hours of sample collection. Hepcidin (Hepcidin-25 [human] EIA kit; Bachem), ferritin (IMx ferritin assay, Microparticle Enzyme Immunoassay; Abbot Laboratories), soluble transferrin receptor (sTfR, Human sTfR ELISA; R&D Systems), serum iron, unsaturated iron binding capacity (UIBC, Ferrozine-based photometry and colorimetry; Hitachi 911 automated analyzer), and α
1-antichymotrypsin (ACT, immunoturbidimetry, Cobas Mira Plus Bioanalyzer, Roche) were assayed according to the manufacturers’ instructions from plasma samples stored at -80°C. Transferrin saturation (TSAT) was calculated from plasma iron and UIBC (TSAT = [plasma iron/ (UIBC + plasma iron)] X 100)
27 (link). Hepcidin/ferritin and TSAT/hepcidin ratios were also calculated
28 (link). Giemsa-stained thick and thin blood films were examined for
Plasmodium falciparum and other
Plasmodium species at the start and end of the malaria season.
1-antichymotrypsin (ACT, immunoturbidimetry, Cobas Mira Plus Bioanalyzer, Roche) were assayed according to the manufacturers’ instructions from plasma samples stored at -80°C. Transferrin saturation (TSAT) was calculated from plasma iron and UIBC (TSAT = [plasma iron/ (UIBC + plasma iron)] X 100)
27 (link). Hepcidin/ferritin and TSAT/hepcidin ratios were also calculated
28 (link). Giemsa-stained thick and thin blood films were examined for
Plasmodium falciparum and other
Plasmodium species at the start and end of the malaria season.
4
Cross-Sectional Study of Malaria, Anemia, and Hepcidin in Gambian Children
5
Venous Blood Analysis for Iron Status
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Venous blood samples were obtained from a forearm vein for assay of hemoglobin (Hb), hematocrit (Hct), mean cell volume (MCV), serum iron, serum ferritin, serum transferrin, transferrin saturation, plasma hepcidin, and plasma C‐reactive protein (CRP). Blood was distributed into tubes containing EDTA (EDTA BD Vacutainer®, BD) or micronized silica particles (SST BD Vacutainer®, BD) for immediate hematology and biochemistry analysis; plasma was prepared by centrifugation at 3000g and immediately frozen at –80°C for later analysis of hepcidin by enzyme‐linked immunosorbent assay (ELISA, Hepcidin‐25 human‐ EIA Kit, Bachem). The ELISA analysis was in duplicate and followed the guidelines of the manufacturer.
If a participant's CRP was elevated above the normal range for the assay (>8 mg/L), hematological indices from that visit were excluded from analysis in case there was an intercurrent mild inflammatory illness that might have temporarily disturbed iron homeostasis (Feelders et al.,1998 ; Gabay and Kushner, 1999 ; Ganz, 2003 ).
If a participant's CRP was elevated above the normal range for the assay (>8 mg/L), hematological indices from that visit were excluded from analysis in case there was an intercurrent mild inflammatory illness that might have temporarily disturbed iron homeostasis (Feelders et al.,
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