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Title: VIROLOGY, UNIVERSITY PUTRA MALAYSIA Examiners Committee Approved
Keywords: Molecular Immunology
Issue Date: 2013
Publisher: Alneelain University
Abstract: Abstract: Background: Recombinant antibody cloning and phage display technologies proved very efficient in producing and isolating single-chain antibodies (scFv), especially against antigen of interest, which is MCF-7 breast cancer cell line. Aim: The main objectives of this study are to construct single chain variable fragment (scFv) towards MCF-7 breast cancer cells and to characterize scFv antibodies that interacts with MCF-7 breast cancer cells line. Materials and Methods: Therefore, phage display technologies were used to produce single- chain antibodies (scFv) against (MCF-7) breast carcinoma cell line. The starting material was the mouse B cell hybridoma line C3A8, which generates a monoclonal antibody against breast cancer cells (MCF-7). The integrated cloning, screening, and phage display system allowed to obtain a scFv sequences derived from the hybridoma C3A8 cell line. The extended primer set can incorporate the entire mouse VH and V1, sequences collected in the Kabat database. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were sub-cloned into HB2l51 host strain. The expressed product was characterized by westem blot and indirect ELISA. The scFv gene was then cloned into nova-blue host strain for cloning purposes and then into origami DE host strain for further characterization. The purified single-chain antibody expressed in origami DE3 was purified using Immobilized Metal Affinity Chromatography (IMAC). The purified scFv protein was characterized using westem blot, flow Cytometery and immunofluorescence tests. Bioinformatics tools was also used and databases such as BLASTalignment search tool), GenBank, PDB (protein databank), KABAT numbering, SWISS- MODEL and Insight II to gain specific functional insights into scFv anti-MCF-7. VH and VL chains models were evaluated using Verify3D, ERRAT and Ramchandran plot methods of evaluation. Results: A 32-kDa band was observed on the blotting membrane when scFv antibody probed with an anti-his tag monoclonal antibody. In addition, scFv protein was characterized and showed specific binding toward MCF-7 cells line. A band of 68kD was appeared in nitrocellose membrane when westem blot test was used. Further, immuno-fluorescence test clearly proved that the scFv recognized the MCF-7 antigen epitopes, which is localized in MCF-7 nuclear. Moreover, 53% of the cells numbers were bound to scFv protein as measured by flow cytometery analysis. Finally, the predicted stmctures of heavy and light chains were connected with peptide linker to build the full scFv protein structure. The predicted structures of heavy and light chains were found to be acceptable according to the structure evaluation methods and were used to predict conformational and sequential epitopes. Discussion and Conclusions: Herein the recombinant antibody technology is a rapid and efiective approach to the development of the next generation of cancer diagnosis and therapy antibodies.
Appears in Collections:PHD theses : Science

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