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.
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