Poon Kwok Man. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 118-123). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.iv / DECLARATION --- p.vi / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xv / ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1. --- Antibody structure and diversity --- p.1 / Chapter 1.2. --- Antibody genes --- p.5 / Chapter 1.3. --- The antibody response to phosphorylcholine --- p.10 / Chapter 1.3.1. --- Group I antibodies --- p.11 / Chapter 1.3.2. --- Group II antibodies --- p.14 / Chapter 1.3.3. --- Fine specificity of group I antibodies --- p.14 / Chapter 1.4. --- Anti-phosphorylcholine antibody structure --- p.15 / Chapter 1.5. --- Recombinant antibody --- p.22 / Chapter 1.5.1. --- Phage biology --- p.24 / Chapter 1.5.2. --- Phage-displayed antibodies --- p.29 / Chapter 1.5.3. --- Helper phage --- p.32 / Chapter 1.6. --- Objectives and scope of study --- p.34 / Chapter CHAPTER 2 --- METHODOLGY / Chapter 2.1. --- Antibody --- p.41 / Chapter 2.1.1. --- Hybridoma culture --- p.41 / Chapter 2.1.2. --- Production of antibody by induction of ascitic fluid --- p.41 / Chapter 2.1.3. --- Antibody purification --- p.41 / Chapter 2.1.3.1. --- Ammonium sulfate precipitation --- p.42 / Chapter 2.1.3.2. --- Affinity purification by Protein A-sepharose --- p.42 / Chapter 2.1.4. --- Production of Fab fragment by papain digestion --- p.43 / Chapter 2.2. --- Antigens --- p.43 / Chapter 2.2.1. --- Preparation of TsAg form infected ICR mouse --- p.44 / Chapter 2.2.2. --- Purification of Trichinella spairalis PC antigen --- p.44 / Chapter 2.2.2.1. --- Preparation of Mab2 affinity column --- p.44 / Chapter 2.2.2.2. --- Purification of TsAg --- p.45 / Chapter 2.2.3. --- Preparation of PC-HSA --- p.45 / Chapter 2.2.3.1. --- Preparation of p-diazonium phenylphosphorylcholine (DPPC) --- p.45 / Chapter 2.2.3.2. --- Conjugation of PC to HSA --- p.45 / Chapter 2.2.4. --- Commercial available antigens --- p.46 / Chapter 2.2.4.1. --- Pneumovax® 23 --- p.46 / Chapter 2.2.4.2. --- Lipopolysaccharide --- p.46 / Chapter 2.2.5. --- Standardization of PC-antigens --- p.46 / Chapter 2.3. --- Cloning of Mab2-scFv into phage display form --- p.47 / Chapter 2.3.1. --- Total RNA extraction --- p.50 / Chapter 2.3.2. --- cDNA synthesis --- p.50 / Chapter 2.3.3. --- Heavy chain variable region gene amplification --- p.51 / Chapter 2.3.4. --- Light chain variable region gene amplification --- p.51 / Chapter 2.3.5. --- Joining of heavy and light chain gene with linker --- p.52 / Chapter 2.3.6. --- Ligation of scFv gene with pCANTAB-5E vector --- p.52 / Chapter 2.3.7. --- Transformation --- p.53 / Chapter 2.3.7.1. --- E.coli strains --- p.53 / Chapter 2.3.7.2. --- E.coli cell preparation for electroporation --- p.54 / Chapter 2.3.7.3. --- Electroporation --- p.54 / Chapter 2.3.7.4. --- Competent E.coli preparation by CaCl2 --- p.55 / Chapter 2.3.7.5. --- Heat shock --- p.55 / Chapter 2.4. --- Expression of phage display scFv --- p.55 / Chapter 2.5. --- Enrichment and screening of Mab2-scFv phage --- p.56 / Chapter 2.5.1. --- Biopanning --- p.56 / Chapter 2.5.2. --- Restricition fragment analysis --- p.58 / Chapter 2.5.3. --- PCR screening --- p.58 / Chapter 2.5.4. --- DNA sequencing --- p.58 / Chapter 2.5.4.1. --- Manual sequencing --- p.58 / Chapter 2.5.4.2. --- Auto sequencing --- p.59 / Chapter 2.6. --- Mutagenesis --- p.59 / Chapter 2.6.1. --- Preparation of Uracil containing ssDNA --- p.60 / Chapter 2.6.2. --- Phosphorylation of mutagenic oligonucleotide --- p.60 / Chapter 2.6.3. --- Hybridization and secondary strand synthesis...…… --- p.60 / Chapter 2.6.4. --- Transfection and screening of mutants --- p.61 / Chapter 2.7. --- Expression of soluble scFv-E-tag --- p.61 / Chapter 2.7.1. --- SDS-PAGE analysis --- p.62 / Chapter 2.7.2. --- Anti-E-tag ELISA --- p.62 / Chapter 2.8. --- ELISA binding assay --- p.63 / Chapter 2.8.1. --- Specificity of Mab2 antibody Fab --- p.63 / Chapter 2.8.1.1. --- Carrier specifcity assay --- p.63 / Chapter 2.8.1.2. --- Free hapten inhibition assay --- p.64 / Chapter 2.8.2. --- Specificity of the scFv --- p.64 / Chapter 2.8.2.1. --- Antigen binding assay --- p.65 / Chapter 2.8.2.2. --- Free hapten inhibition assay --- p.65 / Chapter 2.8.2.3. --- Inhibition on Ts2 and Mab2 antibody assay --- p.65 / Chapter 2.9. --- Affinity assay --- p.66 / Chapter 2.10. --- Mutants analysis --- p.66 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1. --- Cloning VH and VL gene of Mab2 into scFv --- p.67 / Chapter 3.1.1. --- Amplification of variable region of H and L chain --- p.67 / Chapter 3.1.2. --- Biopanning --- p.70 / Chapter 3.1.3. --- Genetic composition of isolated clones --- p.70 / Chapter 3.2. --- Mutagenesis --- p.84 / Chapter 3.3. --- Expression and characterisation of wild-type scFv --- p.88 / Chapter 3.3.1. --- ScFv soluble protein --- p.88 / Chapter 3.3.2. --- Phage displayed scFv --- p.91 / Chapter 3.3.3. --- Standardization of PC antigens --- p.91 / Chapter 3.3.4. --- Binding acticity of scFv --- p.94 / Chapter 3.3.4.1. --- Influence of the avidity on carrier specificity binding --- p.96 / Chapter 3.4. --- Antigen specificity --- p.99 / Chapter 3.4.1. --- Free hapten inhibiton --- p.99 / Chapter 3.4.2. --- Inhibition on the binding of Ts2 --- p.102 / Chapter 3.4.3. --- Binding affinity --- p.104 / Chapter 3.5. --- Binding activities of mutants --- p.106 / Chapter CHAPTER 4 --- GENERAL DISCUSSION --- p.109 / REFERENCE --- p.118

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 10 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept., 2009). Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 16, 2010). Includes bibliographical references.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination. / Dissertation