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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Date
2015-09
Authors
QIUTING, LOH
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Abstract
The 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.
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Keywords
Comparison Of Fab And Scfv Phage Display Efficiency , Application Of Best Format For The Development Of A Novel Dna G-Quadruplex Based Immunoassay