Comparison Of Fab And Scfv Phage Display Efficiency: Application Of Best Format For The Development Of A Novel Dna G-Quadruplex Based Immunoassay

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dc.contributor.authorQIUTING, LOH
dc.date.accessioned2016-05-12T07:40:42Z
dc.date.available2016-05-12T07:40:42Z
dc.date.issued2015-09
dc.description.abstractThe conventional diagnostic platforms are largely dependent on the availability of murine monoclonal antibodies and are designed to be used on platforms such as immunoassays and lateral flow. One of the main issues associated with conventional diagnostic methods is the sensitivity and availability of biomolecules. In designing a diagnostic platform, there are two main components that have to be considered. It is mainly the development of bio-binders and a reporter system. In the case of a biobinder development, recombinant antibodies were selected using phage display technology. A comparison between the scFv (single-chain fragment variable) and Fab (fragment of antigen binding) was carried out in terms of development, identification and production. The main challenge in using Fab fragments is the complexity of the disulphide bond formation that normally results in lower display efficiencies of Fab fragments during phage display. Therefore, in this study, molecular chaperones such as DsbA and DsbC that were encoded by the helper plasmid, pTUM4 were introduced in order to improve the disulphide bond formation to aid display efficiencies during phage display. A modified version of the panning using the helper plasmid system was evaluated and used for the development of antigen specific Fab binders. On the other hand, panning using scFv library was also carried out. Although the presentation of Fab was improved, the scFv format was advantageous in the development process making it more ideal for immunoassay development. Conventional immunoassays often uses enzyme-conjugated secondary antibody as the reporter which sometimes suffers from sensitivity, stability, durability and the costs of the assay. Subsequently, new detection methods other than antibody-enzyme conjugates have been developed to counter these issues. Many have incorporated nucleic acid amplification methods into immunoassays with an amplification step for increased sensitivity. The recombinant scFv binder was successfully adapted to a novel immunoassay method which uses G-quadruplexes DNAzymes as the reporter system. The immuno-quadruplex priming amplification (IQPA) method uses self-dissociating G-quadruplex structures to function as DNAzymes to generate a colorimetric readout. Optimization of the assay provides a proof-of-concept of the platform for immunoassay development. In the direct IQPA, the limit of detection was found to be 0.5 μM, nevertheless, in competitive IQPA, it can detect as low as 1 fM of antigen. In conclusion, the fusion of recombinant antibody technology and DNA nanotechnology provides an alternative avenue for the development of novel diagnostic platforms with improved sensitivities and ease of use.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/2035
dc.subjectComparison Of Fab And Scfv Phage Display Efficiencyen_US
dc.subjectApplication Of Best Format For The Development Of A Novel Dna G-Quadruplex Based Immunoassayen_US
dc.titleComparison Of Fab And Scfv Phage Display Efficiency: Application Of Best Format For The Development Of A Novel Dna G-Quadruplex Based Immunoassayen_US
dc.typeThesisen_US
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