Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin.
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.
Obzansky
D M
DM
E.I. du Pont de Nemours & Co., Inc., Medical Products, Wilmington, DE 19898.
Rabin
B R
BR
Simons
D M
DM
Tseng
S Y
SY
Severino
D M
DM
Eggelte
H
H
Fisher
M
M
Harbron
S
S
Stout
R W
RW
Di Paolo
M J
MJ
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*
D013972:Thyrotropin; D006861:Hydrogen Peroxide