title: Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin.
abstract: A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active holo D-AAO oxidizes D-proline to produce hydrogen peroxide, which is quantified by the horseradish peroxidase-mediated conversion of 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to a colored product. The FADP-based enzyme amplification cascade has been used in a novel releasable linker immunoassay (RELIA) to quantify thyrotropin (TSH). In the assay, TSH is first captured onto antibody-coated chromium dioxide particles. After formation of an antibody-TSH sandwich with a dethiobiotinylated second antibody, the complex is reacted with a streptavidin-ALP conjugate. Biotin is then used to release the conjugate into solution, and ALP is quantified in an automated version of the FADP-based amplification cascade on the aca discrete clinical analyzer (Du Pont). The sensitivity of the colorimetric RELIA assay for TSH (less than 0.1 milli-int. unit/L) is comparable with that of fluorometric assays. This technology provides a way to adapt to the aca high-sensitivity immunoassays for a wide range of analytes via colorimetric detection.
authors.affiliation: E.I. du Pont de Nemours & Co., Inc., Medical Products, Wilmington, DE 19898.
authors.forename: D M
authors.initials: DM
authors.lastname: Obzansky
authors.forename: B R
authors.initials: BR
authors.lastname: Rabin
authors.forename: D M
authors.initials: DM
authors.lastname: Simons
authors.forename: S Y
authors.initials: SY
authors.lastname: Tseng
authors.forename: D M
authors.initials: DM
authors.lastname: Severino
authors.forename: H
authors.initials: H
authors.lastname: Eggelte
authors.forename: M
authors.initials: M
authors.lastname: Fisher
authors.forename: S
authors.initials: S
authors.lastname: Harbron
authors.forename: R W
authors.initials: RW
authors.lastname: Stout
authors.forename: M J
authors.initials: MJ
authors.lastname: Di Paolo
mesh_terms: D004796:Clinical Enzyme Tests / Q000379:methods*; D003124:Colorimetry; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis; D007118:Immunoassay; D007700:Kinetics; D010084:Oxidation-Reduction; D010766:Phosphorylation; D013972:Thyrotropin / Q000032:analysis*
chemical_list: D013972:Thyrotropin; D006861:Hydrogen Peroxide