Major factors limiting sensitivity of sandwich enzyme immunoassay for ferritin, immunoglobulin E, and...

By using IgG-coated polystyrene balls and beta-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 beta-D-galactosidase-labelled Fab' used. (1) Their sensitivities were enhanced 10 to 50-fold by using affinity-purified Fab' labelled with beta-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 millimicronU (5.7 amol) per tube, respectively.