by Frankie Kar-Ming Wong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 101-107). / Acknowledgements --- p.IV / Abstract --- p.V / Abbreviations --- p.VI / Chapter Chapter 1 --- Introduction to Lipoprotein and Apolipoprotein --- p.1 / Chapter 1.1 --- Lipoprotein structure and classification --- p.1 / Chapter 1.2 --- Apolipoprotein A-I and B100 --- p.1 / Chapter 1.2.1 --- Apolipoprotein A-I (apoA-I) --- p.1 / Chapter 1.2.2 --- Apolipoprotein B100 (apoB100) --- p.3 / Chapter 1.2.3 --- Biological functions of apolipoprotein --- p.4 / Chapter 1.3 --- Evidence linking apoA-I and B100 with atherosclerosis --- p.4 / Chapter 1.4 --- The roles of apoA-I and B100 in the development of atherosclerosis --- p.6 / Chapter 1.5 --- Measurement of human serum lipoproteins as an assessment of risk for coronary heart disease (CHD) --- p.8 / Chapter 1.6 --- Aims of this study --- p.10 / Chapter Chapter 2 --- Purification of ApoA-I and B100 and Production of Polyclonal Antibodies --- p.12 / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.1.1 --- Purification of apoA-I and B100 from human serum --- p.12 / Chapter 2.1.2 --- Immunization for polyclonal antibodies production against apoA-I and B100 --- p.14 / Chapter 2.1.3 --- Antibody purification --- p.15 / Chapter 2.1.3.1 --- Ammonium sulfate precipitation --- p.17 / Chapter 2.1.3.2 --- DEAE and QEAE Sepharose --- p.17 / Chapter 2.1.3.3 --- Protein A and Protein G --- p.17 / Chapter 2.1.3.4 --- Affinity chromatography --- p.18 / Chapter 2.2 --- Methods --- p.20 / Chapter 2.2.1 --- Purification of HDL and LDL --- p.20 / Chapter 2.2.2 --- Purification of apolipoproteins --- p.22 / Chapter 2.2.3 --- Immunization of rabbit with apoA-I and B100 --- p.23 / Chapter 2.2.4 --- Enzyme-linked immunosorbent assay (ELISA) --- p.24 / Chapter 2.2.5 --- Purification of lipoprotein specific immunoglobulin from antisera --- p.25 / Chapter 2.2.5.1 --- Salt fractionation --- p.25 / Chapter 2.2.5.2 --- Purification of immunoglobulin by Protein A affinity chromatography --- p.25 / Chapter 2.2.5.3 --- Isolation of specific antibody by lipoprotein-coupled affinity chromatography --- p.26 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Purification of apoA-I and B100 --- p.27 / Chapter 2.3.2 --- Purification of immunoglobulins from rabbit anti-apolipoprotein sera --- p.32 / Chapter 2.4 --- Discussion --- p.38 / Chapter Chapter 3 --- Production of monoclonal antibodies against apoA-I and B100 --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.1.1 --- What is monoclonal antibody? --- p.48 / Chapter 3.1.2 --- The basic methodology --- p.49 / Chapter 3.1.2.1 --- Immunization of host --- p.49 / Chapter 3.1.2.2 --- Cell lines required for fusion --- p.49 / Chapter 3.1.2.3 --- Fusion --- p.51 / Chapter 3.1.2.4 --- Selection of hybrids --- p.52 / Chapter 3.1.2.5 --- Screening assay --- p.54 / Chapter 3.1.2.6 --- Cloning --- p.54 / Chapter 3.1.2.7 --- Bulk production of monoclonal antibody --- p.55 / Chapter 3.1.2.8 --- Monoclonal antibody purification --- p.55 / Chapter 3.2 --- Methods --- p.55 / Chapter 3.2.1 --- Immunization of mice with apoA-I and apoB100 --- p.55 / Chapter 3.2.2 --- Preparation before fusion --- p.58 / Chapter 3.2.2.1 --- Preparation of tissue culture working solutions --- p.58 / Chapter 3.2.2.2 --- Preparation of spleen cells --- p.59 / Chapter 3.2.2.3 --- Preparation of myeloma cells --- p.60 / Chapter 3.2.3 --- Fusion --- p.60 / Chapter 3.2.4 --- Screening assay for positive clones --- p.61 / Chapter 3.2.5 --- Limiting dilution cloning --- p.61 / Chapter 3.2.6 --- Determination of isotype --- p.62 / Chapter 3.2.7 --- Cryopreservation of myeloma and established hybridoma cell lines --- p.62 / Chapter 3.2.7.1 --- Freezing cells --- p.62 / Chapter 3.2.7.2 --- Thawing cells --- p.63 / Chapter 3.2.8 --- Bulk production of monoclonal antibodies from ascites --- p.63 / Chapter 3.2.9 --- Purification of monoclonal antibodies from ascites --- p.63 / Chapter 3.2.10 --- Western blot analyses of the monoclonal antibodies --- p.64 / Chapter 3.2.11 --- Iodination of apolipoproteins --- p.64 / Chapter 3.2.12 --- Binding of the monoclonal antibody to iodinated apolipoprotein --- p.65 / Chapter 3.2.13 --- Competitive displacement analyses --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Development of monoclonal antibodies --- p.66 / Chapter 3.3.2 --- Purification of monoclonal antibody from ascites --- p.69 / Chapter 3.3.3 --- Western blotting analyses of AB6 and BE8 --- p.69 / Chapter 3.3.4 --- Monoclonal antibody titration curve for apolipoproteins by radioimmunoassays --- p.75 / Chapter 3.3.5 --- Competitive displacement analysis of AB6 and BE8 --- p.75 / Chapter 3.4 --- Discussion --- p.79 / Chapter Chapter 4 --- Enzyme-linked immunosorbent assay (ELISA) for ApoA-I --- p.84 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.1.1 --- Alkaline phosphatase (ALP) --- p.84 / Chapter 4.1.2 --- Conjugation methods --- p.85 / Chapter 4.1.3 --- Design of the immunoassay format --- p.87 / Chapter 4.1.4 --- Modified solid-phase: Protein A antibody-capture ELISA (PACE) --- p.87 / Chapter 4.2 --- Materials and Methods --- p.90 / Chapter 4.2.1 --- Conjugation of AB6 with maleimide activated alkaline phosphatase --- p.90 / Chapter 4.2.2 --- Titration curve of AB6-ALP conjugate --- p.90 / Chapter 4.2.3 --- Calibration curve of apoA-I sandwich ELISA --- p.91 / Chapter 4.2.4 --- Measurement of apoA-I by Protein A antibody-capture ELISA --- p.91 / Chapter 4.3 --- Results --- p.92 / Chapter 4.3.1 --- Characterization of AB6-ALP conjugate --- p.92 / Chapter 4.3.2 --- Calibration curve for the measurement of apoA-I --- p.92 / Chapter 4.4 --- Discussion --- p.95 / Chapter Chapter 5 --- General Conclusions --- p.99 / References --- p.101
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322727 |
| Date | January 1997 |
| Contributors | Wong, Frankie Kar-Ming., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xi, 107 leaves : ill. ; 30 cm. |
| Rights | Use 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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