Improved sensitivity in homogeneous enzyme immunoassays using a fluorogenic macromolecular substrate: An assay for serum ferritin

A new highly sensitive nonseparation enzyme immunoassay for human serum ferritin is described. Reagents include a β-galactosidase-ferritin conjugate, sheep anti-ferritin, anti-sheep IgG, and dextran-linked β-galactosylumbelliferone as enzyme substrate. The method is based on inhibition of enzyme activity when anti-ferritin binds to the enzyme-ferritin conjugate. Ferritin in the sample and enzyme-labeled ferritin compete for a limited quantity of antiferritin. The enzyme activity of the reaction mixture is directly related to the ferritin content of the sample. Some patients' samples caused strong interference in the assay due to the presence of antibody to β-galactosidase. Several ways of eliminating the interference are presented. When measures were adopted to suppress sample interference, the assay results correlated well with those of other immunoassay methods.