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Studies on the nature and applications of antibody-derived binding-site molecules

McMahon, Michael John (1999) Studies on the nature and applications of antibody-derived binding-site molecules. PhD thesis, Dublin City University.

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.
Item Type:Thesis (PhD)
Date of Award:1999
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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