Novel and sensitive noncompetitive (two-site) enzyme immunoassay for haptens with amino groups

A novel and sensitive noncompetitive (two-site) enzyme immunoassay for haptens with amino groups is described. L-Thyroxine (T4) was used as a model hapten. T4 was indirectly biotinylated with glutathione as spacer between T4 and biotin molecules and trapped onto anti-(T4-bovine serum albumin) IgG-coated polystyrene balls. After washing the polystyrene balls to eliminate unreacted biotin and other biotinylated substances, biotinylated T4 was eluted from the polystyrene balls with HCl and was reacted with anti-(T4-bovine serum albumin) Fab'-horseradish peroxidase conjugate. The complex formed was trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. The detection limit of T4 was 78 fg (0.1 fmol)/tube, which was 50-fold lower than the limit by competitive enzyme immunoassay using the same antibody and T4-peroxidase conjugate. This noncompetitive enzyme immunoassay was applied to the measurement of total T4 in serum. Serum levels of total T4 in 10 healthy subjects aged 25-40 yr were 94 +/- 13 (SD) micrograms/L (range, 78-114). The principle of this noncompetitive enzyme immunoassay may allow it to measure other haptens with amino groups more sensitively than can competitive immunoassays.