| Popis: |
Immunoassay techniques for the detection of infectious agents, hormones and cellular antigens have been investigated to improve assay performance for sensitivity, detection limits, assay protocols and incubation times. Such improvements included using high affinity monoclonal antibodies for capture and detection, allowing measurement of smaller sample volumes and shorter incubation times. Enzyme labels including horseradish peroxidase, alkaline phosphatase and luciferase were evaluated using chemiluminescence substrate systems for increased sensitivity. New two-dimensional, photon imaging detectors were required to develop fully these luminescent assays. In this thesis the imaging photon detector (IPD), cooled charge-coupled device (CCD) and intensified CCD, which were adapted for luminescence detection, are described. This adaptation required specially designed optics and sample presentation units, together with calibration and analysis software to achieve the required sensitivity, dynamic range (>104), and precise quantitative measurements. Applications which were developed using this luminescence imaging technique were: enzyme assays for ATP detection using firefly luciferase and luciferin (range 10-11 to 10-16 moles ATP), and for 4-methylumbelliferone; enzyme immunoassays for infectious diseases and hormones including Hepatitis B and AFP; multiple, simultaneous detection of groups of diagnostically related assays. A rapid luminescence assay for hCG was developed in a new format using microbeads and a membrane for support, which gave good correlation with RIA and required only 10 minutes for sample incubation. Further applications investigated included luminescent Western blots for protein detection and DNA dot blots, for which the imaging technique allowed quantitative analysis with improved sensitivity over a wide dynamic range compared with film. Detection limits for an alkaline phosphatase labelled dot blot assay for DNA (pBR322) were measured down to a very low level (70 fg of DNA), with sensitivity limited by assay membrane background. Reporter gene assays, employing firefly and bacterial luciferase, were used to detect gene expression in mammalian and bacterial cells. These reporter gene assays are rapid, sensitive and selective and were demonstrated in single mammalian cells with a luciferase-vaccinia virus recombinant. Luminescence imaging has been shown in this thesis to be a new quantitative technique for the measurement of luminescent and fluorescent processes in a wide range of biomedical applications, which offers an advance in analytical and diagnostic methods in the fields of cellular and molecular biology and medicine. |
