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Production and characterization of an anti-telomerase monoclonal antibody.

Xu, Guolin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-128). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGEMENTS --- p.IV / LIST OF FIGURES --- p.VII / ABBBREVIATIONS --- p.X / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Antigens --- p.1 / Chapter 1.1.1 --- Preamble --- p.1 / Chapter 1.1.2 --- Types of antigen --- p.1 / Chapter 1.1.3 --- Autoantigens --- p.2 / Chapter 1.1.4 --- Telomerase is an important autoantigen --- p.6 / Chapter 1.2 --- Antibodies --- p.12 / Chapter 1.2.1 --- Preamble --- p.12 / Chapter 1.2.2 --- Ig structure --- p.12 / Chapter 1.2.3 --- Ig synthesis --- p.13 / Chapter 1.2.4 --- Immunoglobulin isotypes --- p.15 / Chapter 1.2.5 --- Monoclonal antibodies (mAb) --- p.17 / Chapter 1.2.6 --- Autoantibodies --- p.20 / Chapter 1.2.7 --- Telomerase detection and antibodies to telomerase --- p.22 / Chapter 1.3 --- Object and Scope of Study --- p.24 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Animals --- p.31 / Chapter 2.1.2 --- Antibodies --- p.31 / Chapter 2.1.3 --- Primers --- p.31 / Chapter 2.1.4 --- Culture media and reagents --- p.32 / Chapter 2.1.5 --- Chemicals and enzymes --- p.32 / Chapter 2.1.6 --- Miscellaneous chemicals --- p.33 / Chapter 2.1.7 --- Commercial kits --- p.33 / Chapter 2.1.8 --- Instruments --- p.33 / Chapter 2.1.9 --- Buffers --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Cells and cell culture --- p.35 / Chapter 2.2.2 --- Polymerase chain reaction (PCR) --- p.36 / Chapter 2.2.3 --- Cloning of C-terminal gene fragment to pGEX cloning vector --- p.36 / Chapter 2.2.4 --- Detection of antibody activity by ELISA --- p.37 / Chapter 2.2.5 --- Histochemical Staining --- p.38 / Chapter 2.2.6 --- Hybridoma production --- p.40 / Chapter 2.2.7 --- Protein analysis --- p.43 / Chapter 2.2.8 --- Flow cytometry --- p.46 / Chapter 2.2.9 --- Animal handling --- p.47 / Chapter 2.2.10 --- Statistical analysis --- p.48 / Chapter CHAPTER THREE --- PRELIMINARY STUDIES USING THE ANTI-N-TERT-TELOMERASE MAB DERIVED FROM HYBRIDOMA 476 --- p.49 / Chapter 3.1 --- Preamble --- p.49 / Chapter 3.2 --- Hybridoma 476 cells can be stained by the labeled recombinant N-TERT antigen --- p.50 / Chapter 3.3 --- Mouse spleen cells can also be stained by biotin-labeled N-TERT antigen --- p.52 / Chapter 3.4 --- Discussion --- p.53 / Chapter CHAPTER FOUR --- PRODUCTION OF MONOCLONAL ANTIBODIES TO C-TERT --- p.63 / Chapter 4.1 --- Preamble --- p.63 / Chapter 4.2 --- Construction of C-TERT expression vector --- p.63 / Chapter 4.3 --- Expression and purification of recombinant human C-terminal telomerase antigen --- p.64 / Chapter 4.4 --- Immunization of Balb/c mice with C-TERT-GST --- p.65 / Chapter 4.5 --- Generation of hybridomas to C-TERT --- p.65 / Chapter 4.6 --- Identification and selection of reactive clones --- p.65 / Chapter CHAPTER FIVE --- CHARACTERIZATION OF MAB A63 --- p.71 / Chapter 5.1 --- Preamble --- p.71 / Chapter 5.2 --- Characterization of mAb A63 by ELISA --- p.71 / Chapter 5.3 --- Characterization of mAb A63 by Western blotting analysis --- p.73 / Chapter 5.4 --- Characterization of mAb A63 by immuno-histochemical staining --- p.73 / Chapter 5.5 --- mAb A63 can also stain fish telomerase and human placenta --- p.75 / Chapter 5.6 --- mAb A63 can also stain telomerase in human tumors --- p.76 / Chapter 5.7 --- Hybridoma A63 can produce ascites fluid --- p.76 / Chapter 5.8 --- Discussion --- p.77 / Chapter CHAPTER SIX --- GENERAL DISCUSSION --- p.93 / Chapter 6.1 --- Why Enhancing buffer is required for the nuclear staining of hTERT when using mAb 476 or mAb A63 --- p.96 / Chapter 6.2 --- Why hybridoma 476 failed to form ascites while hybridoma A63 succeeded --- p.99 / Chapter 6.3 --- Can IL-6 be used to treat autoimmune diseases? --- p.102 / Chapter 6.4 --- Possible use of monoclonal antibodies in cancer therapy --- p.104 / Chapter 6.5 --- Prospects on study --- p.107 / REFERENCES --- p.111

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326656
Date January 2009
ContributorsXu, Guolin., Chinese University of Hong Kong Graduate School. Division of Chemical Pathology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xi, 128 leaves : ill. ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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