Development of a Highly Sensitive Enzyme Immunoassay for Serum Ferritin and Its Application for Serodiagnosis of Iron Deficiency Anemia

Ferritin is an iron storage protein identified in various tissues such as liver, spleen and bone marrow. Serum ferritin concentration is an excellent indicator of body iron storage, and is closely related to the amount of storage iron in patients with iron deficiency or with iron overload. Another clinical application of serum ferritin measurement is serodiagnosis of malignancies. High values of serum ferritin were found in the patients with leukemia, pancreatic carcinoma, hepatoma and malignant lymphoma. Serum ferritin concentration has generally been estimated using radioimmunoassay (RIA). RIA methods, however, suffer from several disadvantages including expensive reagents, potential health risks from isotopes and the requirement of specialized equipment. Enzyme immunoassay methods (EIA) hitherto reported for serum ferritin using alkaline phosphatase or peroxidase as marker enzymes were less sensitive than RIA methods. We describe in this report a highly sensitive enzyme immunoassay system for serum ferritin with use of affinity-purified anti-ferritin Fab'-β-D-galactosidase complexes, and evaluated its implication for diagnosis of iron deficiency anemia. Polystylene balls were coated with rabbit anti-ferritin IgG which was not purified by affinity chromatography. Affinity-purified IgG was digested to Fab' fragments, and conjugated with β-D-galactosidase in the presence of N-N'-o-phenylenedimaleimide. Using the anti-ferritin IgG coated polystylene balls and antiferritin Fab'-β-D-galactosidase complexes, enzyme linked sandwich assays were carried out and the following results were obtained. (1) The range of ferritin concentration covered by this assay system using 0.1 μl of serum were 0.9-4,555 ng/ml. When 10 μl of serum was used, even 9 pg/ml of ferritin was detectable without serum interference. (2) The coefficient of intra-assay variation and inter-assay variation were 5.9-7.8% and 6.4-8.6%, respectively. (3) When ferritin concentration in 76 human sera were estimated by both RIA and EIA, the regression equation and correlation coefficient were Y=0.98 X+24.1 and 0.98, respectively. (4) In minimal amounts of labelled anti-ferritin, our EIA system is approximately 12 times as sensitive as RIA. (5) Very low levels of serum ferritin (3 ng/ml 16 ng/ml) could be detected by our EIA in patients with iron deficiency anemia. These results suggested that our EIA is clinically useful for monitoring serum ferritin, especially in iron deficiency anemia. In our EIA, the reagents are more stable and inexpensive. Simple equipment is ?sufficient for our EIA analysis. Therefore, it is hoped that our EIA system be widely used in clinical laboratories for determining serum ferritin