McMahon, Michael John (1999) Studies on the nature and applications of antibody-derived binding-site molecules. PhD thesis, Dublin City University.
Abstract
The suitability o f in vitro immunisation, for monoclonal antibody (Mab) production, was investigated. A panel o f nine Mabs were produced, and analysed for polyreactivity. Three different assays for polyreactivity were utilised, including measurement of the affinity of each Mab for a small panel of antigens. Using these three assays, eight of the nine Mabs were demonstrably polyreactive. It was concluded, that in vitro immunisation may not be suitable for Mab production.
A second area of interest was the generation o f intrabody libraries, for functional genomic analyses. A PCR-based strategy was developed to convert any ScFv gene, from the Nissim library, to an intrabody gene. As a model, an anti-NIP ScFv gene was converted to an intrabody gene. Cytochemical studies indicated correct cytoplasmic localisation o f this intrabody, but poor expression levels.
The strategy was applied to generate a large intrabody library. A novel PCRcloning methodology was used. This utilised T4 DNA polymerase, and required the creation of a novel vector, referred to as pMK. The cloning strategy proved very successful and a 'one-shot' intrabody library o f 1.3xl07 independent clones was generated.
These studies demonstrate the feasibility o f the strategy, but suggest that further studies are required to improve intrabody stability.
Metadata
Item Type: | Thesis (PhD) |
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Date of Award: | 1999 |
Refereed: | No |
Supervisor(s): | O'Kennedy, Richard |
Uncontrolled Keywords: | Binding sites (Biochemistry); Monoclonal antibody production; In-vitro immunisation |
Subjects: | Biological Sciences > Biotechnology |
DCU Faculties and Centres: | DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology |
Use License: | This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License |
ID Code: | 19062 |
Deposited On: | 30 Aug 2013 11:11 by Celine Campbell . Last Modified 30 Aug 2013 11:11 |
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