Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin
| Autor: | R. W Stout, D. M. Obzansky, D. M. Simons, S. Y. Tseng, S. Harbron, B. R. Rabin, H. EggeLte, M. J. Di Paolo, M Fisher, D. M. Severino |
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| Rok vydání: | 1991 |
| Předmět: | |
| Zdroj: | Clinical Chemistry. 37:1513-1518 |
| ISSN: | 1530-8561 0009-9147 |
| DOI: | 10.1093/clinchem/37.9.1513 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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