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We describe a sensitive ELISA for measuring the H-type subunit of human ferritin. A high detection sensitivity was attained by the use of antibodies from different species and an enzyme-conjugated secondary antibody. It consisted of a sandwich assay using a solid phase coated with a rabbit polyclonal antibody for human ferritin from term placenta and a soluble monoclonal antibody for human H-ferritin, followed by a secondary anti-mouse immunoglobulin (Ig)G conjugated to β-galactosidase. The assay was calibrated with purified recombinant human H-ferritin from  E. coli . The colorigenic chlorophenol red β-D-galactopyranoside and the fluorogenic 4-methyl-umbelliferyl-β-D-galactopyranoside substrates were used with similar outcome. The described method permits the measurement of human H-ferritin at a concentration ranging from 0.1 to 100 μg/l (or 20–20000 pg per 200 μl sample) and is accurate at a concentration as low as 0.3 μg/l. The coefficient of variation of the assay was 6.05–10.3 % and the recovery of H-ferritin added to cell lysates was 105.8 ± 7.52 %. Depending on the H-ferritin content of the cell line tested, only 600 to 60 000 cells of different human cell lines were needed to measure their H-territin content.

clinical chemistry and laboratory medicine

An ELISA for the H-Subunit of Human Ferritin which Employs a Combination of Rabbit Poly- and Mice Monoclonal Antibodies and an Enzyme Labeled Anti-Mouse-IgG