In this research, we developed a wide range of biological tools against two distinct targets from future diagnostic or therapeutic points of view. Firstly, we demonstrate that sporadic canine B cell lymphoma mimics the features of human equivalents which in turn will be advantageous for development of canine as well as human therapeutics. With a comparative oncological approach, here we developed a monoclonal antibody (NCD1.2) against canine CD20 which also binds to its human counterpart. Using flow cytometry and tissue microarray, we show that NCD1.2 binds specifically to canine B cell lymphomas (CD20+) and not T-cell lymphoma (CD20-). We also cloned scFv scaffold by linking variable heavy and light chains from NCD1.2 hybridoma by a serine-glycine linker to see if it was active as a biological tool for future therapeutics. Intriguingly, we obtained two different kappa light chains from a single hybridoma cell (scFv3 and scFv7) after antibody phage display. These scFvs were cloned into mammalian vectors for expression in CHO cells and ADEPT - CpG2 vector for yeast expression to see if the activity of these scFvs was retained. Our data suggests that recombinant anti-CD20 scFv might be a useful tool for bioconjugate directed immunotherapies in comparative medicine. Secondly, in addition to mAbs we also developed peptide aptamers which are seldom described but have become attractive agents that typically target a specific biomolecule of interest. Parkinson’s disease (PD) is characterized by formation of lewy bodies (inclusion bodies) in the substantia nigra and the major content of lewy bodies is α-synuclein. To begin with we made recombinant α-synuclein and biophysically characterize this protein under different conditions on a native gel. We also performed Circular dichroism to look at its structure and demonstrate that α helicity could be achieved in presence of SDS. The aim of this project was to develop peptide aptamers, mAbs to α-synuclein, map the binding sites onto the peptides derived from the protein and also on recombinant protein. Further we demonstrated the development of biological tools and their potential ability against α-synuclein in α-synuclein expressing cell lines from future PD therapeutic perspective. Monoclonal antibodies were developed and mAb (3.1) was found to be immunopositive for α-synuclein in parts of kidney and brain. Moreover to estimate the oligomeric state of α-synuclein, we developed assays such as co-transfection of two different constructs i.e. cherry and GFP tagged α-synuclein and Proximity ligation assay to show its self - interaction. Peptide phage display screening (NEB Ph.D. 12 mer library) on recombinant WT α-synuclein was performed to identify aptamers and ultimately novel binding proteins. The peptides were selected based on iteration number and out of the selected panel of peptides; SHACWWDECTGS was found to effectively bind α-synuclein using ELISA. Scanning of peptide GDGNSVLKPGNW (highest iteration number) led to identification of interacting proteins with α-synuclein. Thus in conclusion, we show the validation of different antibody scaffolds and peptide aptamers which could be useful tools from future therapeutics point of view against two well characterized antigens in B cell lymphoma and Parkinson’s disease, respectively.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:712320 |
| Date | January 2015 |
| Creators | Jain, Saurabh Ashok |
| Contributors | Hupp, Ted ; Argyle, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/21117 |
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