Amplified assay of alkaline phosphatase using flavin-adenine dinucleotide phosphate as substrate
| Autor: | Stuart Harbron, Brian R. Rabin, Henny J. Eggelte, Mark Fisher |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
D-Amino-Acid Oxidase
Biophysics Biochemistry Colorimetry (chemical method) chemistry.chemical_compound Ribose Methods Molecular Biology Horseradish Peroxidase chemistry.chemical_classification Chromatography Hydrolysis Substrate (chemistry) Cell Biology Buffer solution Alkaline Phosphatase Chromatography Ion Exchange Enzyme chemistry Luminescent Measurements Chromatography Gel Flavin-Adenine Dinucleotide Phosphorylation Alkaline phosphatase Colorimetry Quantitative analysis (chemistry) |
| Zdroj: | Analytical biochemistry. 206(1) |
| ISSN: | 0003-2697 |
| Popis: | A simple to use, robust, quantitative, and extremelysensitive colorimetric assay for alkaline phosphatase (EC 3.1.3.1), designed to be used as a detection system in diagnostic assays employing antibodies or gene probes, is described. This technology is based on the novel principle of prosthetogenesis, according to which a purpose-designed substrate (a prosthetogen) for a primary analyte-linked enzyme label is hydrolyzed to produce a prosthetic group for a detector enzyme system. The prosthetogen employed here is a derivative of FAD which is phosphorylated at the 3′-position of the ribose ring (FADP), the label enzyme is alkaline phosphatase, and the detector is a D-amino-acid oxidaselhorseradish peroxidase-coupled system. Essentially each turnover of every molecule of alkaline phosphatase produces a molecule of D-amino-acid oxidase for detection. Thus enormous amplification of the initial signal is achieved in short time periods because of the relatively high turnover number of alkaline phosphatase for FADP. The system can be formatted as a stable, preformed, freeze-dried preparation containing all analytical components, which is reconstituted simply by addition of buffer solution. This methodology can quantitate less than 0.1 amol of alkaline phosphatase in 30 min at 25°C using microtiter plates. |
| Databáze: | OpenAIRE |
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