Enzyme immune assay

Peripheral venous blood was collected in tubes without any additives, allowed to coagulate for 60 minutes and centrifuged at 1500g for 12 minutes. The serum was aliquoted into multiple cryo tubes and stored at −80˚C until analysis. The serum concentrations of ferritin, iron, transferrin, total iron-binding capacity, transferrin saturation, soluble transferrin receptor and hepcidin were assessed as markers of iron status. Serum hepcidin and soluble transferrin receptor were measured using commercially available antibodies in an enzyme immune assay (R&D systems, Stillwater, Minnesota, USA), and the rest were measured by a Roche Modular P and E system.
The definition of anaemia was based on gender-specific cut-off values, as recommended by the World Health Organization (WHO) (haemoglobin <13.0 g/dL for men, <12.0 g/dL for women).25 (link) Haematological variables were assessed in fresh EDTA blood by the Sysmex automatic analyser.

ELISA is a qualitative and quantitative method for the detection of immune responses using the specific binding of antigens and antibodies. Briefly, the culture medium in the experimental groups and control group were collected at indicated time and centrifuged at 2,500 rpm for 10 min. The levels of lactate dehydrogenase (LDH) in the supernatants were measured following the instructions from the enzyme immune assay (R&D Systems, Oxon, UK).