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

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 20-20,000 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-ferritin content.