The aim of this project was to demonstrate and develop a confocal readout method for fluorescent immunosorbent assays and investigate its potential advantages in comparison to traditional immunoassays. The key point of a confocal immunosorbent assay is the ability to detect the thin layer of immunoassay in the presence of unbound fluorescent reagents without washing the overlayer. Heterogeneous and homogeneous sandwich immunoassays of human IgG model were demonstrated successfully followed by the use of an empirical decomposition method for quantitative separation of the signals of the thin fluorescent assay layer from the overlayer. The detection limits for the homogeneous and heterogeneous formats of the model were 2.2 and 5.5 ng/ml, respectively. The application of confocal microscopy in kinetic analysis of the antigen-antibody reaction of the human IgG model was studied for homogeneous and heterogeneous formats and two fluorescent labels antibodies (FITC and QDs). The association rates of binding of FITC and QD605 conjugated antibodies to human IgG on prepared surfaces were 5.7×104 and 2.6×104 (M-1s-1) respectively. Confocal detection immunosorbent assay enables the detection of more than one assay along the z-axis. By replacing standard substrates with multiple 30 :m layers of substrates, a high density array of immunosorbent assays was created within a stratified medium. Stacks of up to five modified thin mica substrates of model immunoassays were detected by confocal microscopy. When applied to model assays consisting of human and mouse IgGs on different layers, the z-axis multiplexing of immunosorbant assays was demonstrated. The arrays of multiplexed immunosorbent assays were extended to 3D format by using microcontact printing and the assay density was increased twice by detecting the stack of two substrates which each contained two IgGs assays.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:629226 |
| Date | January 2011 |
| Creators | Ghafari, H. |
| Publisher | Nottingham Trent University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://irep.ntu.ac.uk/id/eprint/57/ |
Page generated in 0.0125 seconds