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

✍ Scribed by Koichiro Tanaka; Takeyuki Kohno; Seiichi Hashida; Eiji Lshikawa

• Publisher: John Wiley and Sons
• Year: 1990
• Tongue: English
• Weight: 442 KB
• Volume: 4
• Category: Article
• ISSN: 0887-8013
• DOI: 10.1002/jcla.1860040312

✦ Synopsis

A novel and sensitive noncompetitive (twosite) enzyme immunoassay for haptens with amino ClrouDs is described. L-Thyroxine (T,)

Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. The detection limit of T, was 78 fg (0.1 fmol)/tube, was used as a model hapten. T, was indirectly biotinylated with glutathione as spacer between T4 and biotin molecules and trapped onto anti-(T,-bovine serum albumin) IgGcoated polystyrene balls. After washing the polystyrene balls to eliminate unreacted biotin and other biotinylated substances, biotinylated T4 was eluted from the polystyrene balls with HCI and was reacted with anti-(T,-bovine serum albumin) Fab'-horseradish peroxidase conjugate. The complex formed was trapped onto streptavidin-coated polystyrene balls.

which was 50-fold lower-than the limit by competitive enzyme immunoassay using the same antibody and T,-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) Fg/L (range, 78-1 14). The principle of this noncompetitive enzyme immunoassay may allow it to measure other haptens with amino groups more sensitively than can competitive immunoassays.