Major Factors Limiting Sensitivity of Sandwich Enzyme Immunoassay for Ferritin, Immunoglobulin E, and Thyroid-Stimulating Hormone

By using IgG-coated polystyrene balls and β-d-galactosidase-labelled Fab', sandwich enzyme immunoassays for human ferritin, immunoglobulin E (IgE), and thyroid-stimulating hormone (TSH) were developed, and their sensitivities were shown to be largely limited by the purity, binding efficiency, and amount of β-d-galactosidase-labelled Fab' used. (1) Their sensitivities were enhanced 10 to 50-fold by using affinity-purified Fab' labelled with β-d-galactosidase. (2) Their sensitivities depended upon the efficiency of specific binding of the labelled Fab' to antigens adsorbed on antibody IgG-coated polystyrene balls. (3) Their sensitivities were improved by using the minimal amount of labelled Fab' that gave a reliable calibration curve. Under optimal conditions the sensitivities obtained for ferritin, IgE, and TSH were 0·09 pg (0·2 amol), 0·48 pg (2·4 amol, 0·2 mIU), and 3 · 2 mμU (5 · 7 amol) per tube, respectively.