TABLE OF CONTENT. Page ACKNOWLEDGEMENTS. iii ENGLISH ABSTRACT THAI ABSTRACT. vii LIST OF TABLES LIST OF FIGURES. xvi ABBREVIATIONS.

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1 x TABLE OF CONTENT ACKNOWLEDGEMENTS ENGLISH ABSTRACT THAI ABSTRACT LIST OF TABLES LIST OF FIGURES ABBREVIATIONS iii iv vii xv xvi xviii CHAPTER I: INTRODUCTION 1.1 Statement of problems Literature reviews Immune system Innate immunity Adaptive immunity Humoral mediated immunity Cell-mediated immunity CD4 molecule Hybridoma technology and monoclonal 24 antibody production Production of anti-cd4 monoclonal 30 antibodies used in this study 1.3 Objectives 31

2 xi CHAPTER II: MATERIALS AND METHODS 2.1 Chemicals, antibodies and instruments used in this study Hybridoma cultivation and culture supernatant production Determination of specific reactivity of anti-cd4 mabs 32 in culture supernatants by COS cell transfection technique COS cell transfection Indirect immunofluorescent assay of transfected 33 COS cells with anti-cd4 mabs 2.4 Large scale production and purification of anti-cd4 mabs Large scale production of anti-cd4 mabs Purification of anti-cd4 mabs by 34 affinity chromatography Determination of the purity of purified 35 anti-cd4 mabs Determination of the reactivity of purified anti-cd4 35 mabs by COS cell transfection technique 2.5 Immunoprecipitation of CD4 protein by anti-cd4 mabs Biotinylation of cell surface molecules Preparation of cell lysates and immunoprecipitation Chemiluminescence detection system Epitope mapping of anti-cd4 mabs by cross blocking analysis 37

3 xii Preparation of FITC-conjugated purified anti-cd4 37 mabs Peripheral blood mononuclear cells (PBMCs) 38 preparation Cross blocking experiment Reactivity of anti-cd4 mabs with CD4 molecule 39 expressed on lymphocytes and monocytes Determination of saturated concentration of 39 anti-cd4 mabs Determination of anti-cd4 mabs reactivity 39 with lymphocytes and monocytes 2.8 Study the effect of anti-cd4 mabs on the regulation of 40 anti-cd3 induced PBMCs proliferation Immobilization of anti-cd3 mab Carboxyfluorescein diacetate succinimidyl ester 40 (CFSE) labeling of PBMCs Proliferation assay Study the effect of anti-cd4 mabs on the regulation of 41 anti-cd3/cd28 induced monocyte-depleted lymphocte proliferation Immobilization of anti-cd3 mab CFSE labeling of monocyte-depleted lymphocytes 41

4 xiii Proliferation assay Study the effect of anti-cd4 mabs on monocyte oxidative 42 burst induction Phorbol myristate acetate (PMA) activation 42 (Control group) Effect of anti-cd4 mabs on monocyte oxidative burst 43 (Experimental group) CHAPTER III: RESULTS 3.1 Specificity of anti-cd4 monoclonal antibodies used in this study Large scale production and purification of anti-cd4 mabs Immunoprecipitation of CD4 molecule using purified 50 anti-cd4 mabs 3.4 Epitope recognition by anti-cd4 mabs Reactivity of anti-cd4 mabs with CD4 molecules expressed 54 on lymphocytes and monocytes Determination of the saturated concentration of anti-cd4 54 mabs for immunefluorescence staining Reactivity of anti-cd4 mabs on CD4 molecules expressed 59 on lymphocyte and monocyte surfaces 3.6 Functional study of CD4 molecule 62

5 xiv Effect of the generated anti-cd4 mabs on anti-cd3 62 induced PBMC proliferation Effect of the generated anti-cd4 mabs on 67 anti-cd3/cd28 induced monocyte-depleted lymphocyte proliferation Effect of the generated anti-cd4 mabs in 70 oxidative burst induction CHAPTER IV: DISCUSSION & CONCLUSION 72 REFERENCES 80 APPENDICES 89 APPENDIX A 90 APPENDIX B 93 APPENDIX C 94 APPENDIX D 95 CURRICULUM VITAE 108

6 xv LIST OF TABLES Table 3.1 Cross blocking analysis of anti-cd4 mabs 53

7 xvi LIST OF FIGURES Figure 1.1 Innate and adaptive immunity Types of adaptive immunity Effector functions of antibodies The induction and effector phases of cell-mediated immunity Effector functions of CD4+ lymphocytes Mechanisms of cytotoxic T lymphocytes Structure of CD4 molecule Function of CD4 molecule CD4 is a receptor for HIV Metabolic pathways of DNA synthesis Pathway of nucleotide synthesis in the present of 28 antifolate drug 1.12 Monoclonal antibody production Immunofluorescence analysis of specific 47 reactivity of anti-cd4 mabs 3.2 SDS-PAGE analysis of the purified anti-cd4 mabs Immunofluorescence analysis of specific reactivity of purified 49 anti-cd4 mabs 3.4 Immunoprecipitation of CD4 molecule by purified 51 anti-cd4 mabs

8 xvii Figure 3.5 Flow cytometric analysis of lymphocytes and monocytes 55 stained with various concentrations of anti-cd4 mabs 3.6 The saturated concentration of anti-cd4 mabs for 57 determination of CD4 molecules expressed on lymphocytes and monocytes 3.7 Flow cytometric analysis of lymphocytes and monocytes 60 by stained with anti-cd4 mabs MT4, MT4/2, and MT4/3 3.8 The reactivity of anti-cd4 mabs MT4, MT4/2, and MT4/3 61 on lymphocytes (A) and monocytes (B) 3.9 Effect of anti-cd4 mabs on anti-cd3 induced 63 lymphocyte proliferation 3.10 Effect of anti-cd4 mabs on lymphocyte proliferation Effect of anti-cd4 mabs on anti-cd3/cd28 68 induced monocyte-depleted lymphocyte proliferation 3.12 Effect of anti-cd4 mabs MT4, MT4/2, and MT4/3 in 71 the induction of monocyte oxidative burst

9 xviii ABBREVIATIONS C Degree Celsius (v/v) volume: volume ratio % Percentage g l APC BSA CD CFSE Microgram Microliter Antigen presenting cell Bovine serum albumin Cluster of differentiation Carboxyfluorescein diacetate succinimidyl ester CMI CTL DHE DMEM Cell-mediated immunity Cytotoxic T lymphocyte Dihydroehidium Dulbecco's Modified Eagle Medium E. coli Escherichia coli EDTA FBS FITC gp HGPRT Ethylenediamine tetraacetic acid Fetal bovine serum Fluorescein isothiocyanate Glycorotein Hypoxanthine guanine phosphoribosyl transferase

10 xix HIV HMI HRP IFN Ig IgG IgM IL IMDM kda L LPS M mab mg MHC ml MW NaCl NaHCO 3 ng OD PAMPs PBMC Human immunodeficiency virus Humoral mediated immunity Horseradish peroxidase Interferon Immunoglobulin Immunoglobulin G Immunoglobulin M Interleukin Iscove's Modified Dulbecco's Medium Kilodalton Liter Lipopolysaccharide Molarity Monoclonal antibody Milligram Major histocompatibility complex Milliliter Molecular weight Sodium chloride Sodium bicarbonate Nanogram Optical density Pathogen-associated molecular pattern Peripheral blood mononuclear cell

11 xx PBS PE PGN ph PKC PMA PMSF RBC SD SDS-PAGE Phosphate buffer saline Phycoerythrin Proteins and peptidoglycans Power of hydrogen Protein kinase C Phorbol myristate acetate Phenylmethyl-sulphonylfluoride Red blood cell Standard deviation sodium dodecyl sulfatepolyacrylamide gel electrophoresis TCR T H TLR TNF TyK T cell receptor Helper T lymphocytes Toll-like receptor Tumor necrosis factor tyrosine kinase

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