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Title: An enzyme amplification cascade for the detection of alkaline phosphatase for use in diagnostic procedures
Author: Fisher, Mark
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1992
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A simple to use, quantitative and extremely sensitive colorimetric assay, designed to be used in a detection system in diagnostic assay systems, is described. The technology of an amplified colorimetric assay, developed by London Biotechnology Limited, is based on a conceptually new principle which is covered by an issued generic patent. The enzyme amplification cascade detects alkaline phosphatase, a widely used enzyme label in immunoassays and gene probes, via dephosphorylation of a novel substrate, FADP, to produce the prosthetic group FAD. This binds stoichiometrically to inactive apo-D-amino acid oxidase, to produce the active holoenzyme which oxidizes D-proline to yield hydrogen peroxide; this in turn is quantitated by a coupled reaction utilizing horseradish peroxidase. The signal-to-noise ratios and sensitivity are enhanced by the kinetics of the amplification cascade. The criteria for the quality control of the reagents used the the cascade system are much more severe than those used in conventional assay systems. Methodologies for the monitoring of contaminants in the production of these materials have been developed. Thus the detection system itself has been used to trouble-shoot the production processes. The primary substrate, FADP, and apo-D-amino acid oxidase have been prepared in high yield and purified to the level required. FADP has been produced with a contamination level of FAD equivalent to 2 ppm and apo-D-amino acid oxidase has been prepared which contains only 10 ppb alkaline phosphatase activity. Evaluation of the technology has demonstrated it to be suitable for use on high quality clinical analyzers; and the methodology has been used in conjunction with the releasable linker technology developed by Du Pont. This system has been used to measure TSH across the entire range of clinical interest.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available