Thesis (M.S.)--Northern Michigan University, 2009. / Includes bibliographical references (leaves 157-171).

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

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Six mouse monoclonal antibodies have been isolated which react against a bacterial fusion protein containing amino acids 364 to 623 of the NS-1 protein of the prototype strain of the Minute Virus of Mice (MVMp). All six were found to be of the IgG class of antibodies; five being IgG₁ and the sixth being IgG₂[formula omitted]. By immunoblot analyses, these antibodies all recognize an 83 kDa protein found only in MVM-infected mouse fibroblast cells, leading to the assumption that they are all NS-1 specific. Further evidence for this assumption is obtained from indirect immunofluorescence studies showing all but one of the mAbs react against a nuclear protein found in MVM-infected cells. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen. For the six monoclonal antibodies, four distinct epitopes were found (A - D). Three were clustered in a 16 amino acid region near the carboxy-terminal of the bacterial fusion protein, while the fourth was slightly more toward the amino-terminal side. Competition ELISAs against a 25 amino acid NS-1 specific peptide confirmed the mapping of the A epitope recognized by the CE10 and AC6 monoclonal antibodies. Also in this thesis, the characterization of a NS-1 fusion protein and a non-fused NS-1 protein expressed in insect cells by recombinant baculoviruses is also described. The latter, a full-length NS-1 protein designated NS-1[formula omitted]ⅽ, was found to be an 84 kDa cytoplasmic protein. This protein was immunoprecipitated by all six monoclonal antibodies. A CE10 monoclonal antibody immunoaffinity column was employed in the single-step purification of NS-1 [formula omitted]c from insect cells. Four elution methods (alkaline, peptide, 6M guanidinium, and acid) were examined and the best purification was obtained using the acid elution. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

A panel of monoclonal antibodies were raised against the 1,4-dihydropyridine sensitive Ca2+ channel of rabbit skeletal muscle. When tested on immunoblot assay of denatured and reduced transverse tubule membranes, four of the antibodies specifically recognized a polypeptide of Mr 140,000. This component co-migrated with the large glycoprotein ?2 subunit of purified Ca2+ channel preparations. On immunoblots of nonreducing gels the antibodies detected a component that migrated more slowly in the gel, with a Mr of 170,000, consistent with the disulphide-linkage of the ?2 subunit to a small component of Mr 30,000. Additionally, three of the antibodies also recognized high molecular weight components of Mr 310,000-330,000 under these conditions. Crossreactive polypeptides of similar apparent molecular weight were detected in immunoblot assays of rabbit heart and brain membranes and of skeletal muscle membranes from different species. Further similarities between the ?2 components of Ca2+ channels from different species were investigated by immunoblot assay, following the limited tryptic digestion of the skeletal muscle membranes. A similar pattern of immunoreactive peptides were detected in each case, suggesting that the ?2 subunits of Ca2+ channels from different species are similar, not only in terms of antibody binding sites but also with respect to similarly positioned trypsin cleavage sites. The extent of glycosylation of the ?2 component was investigated using enzymatic and chemical deglycosylation techniques. Chemical deglycosylation resulted in a core polypeptide of Mr 105,000, consistent with a carbohydrate content of approximately 25%. Enzymatic treatments, although insufficient to completely deglycosylate the ?2 component, reduced the maximal 1,4-dihydropyridine binding capacity of transverse tubule membranes by 73-77%. The co-development of the ?2 subunit with 1,4- dihydropyridine binding activity was shown in rat skeletal muscle. These results indicate that the '2 subunit is an integral structural component of the 1,4-dihydropyridine sensitive Ca2+ channel.

No description available.

A physiologic model for phencyclidine disposition in the rat was established. This model was able to accurately predict phencyclidine disposition in most rat tissues. Physiologic models are based on actual physiologic, anatomic, and biochemical considerations. As a result, these models can be used to predict drug disposition under conditions of altered physiology or anatomy. This aspect of physiologic modeling was tested in the present study by examining the ability of the model to predict phencyclidine plasma disposition in dog and man. The model developed in this study was able to accurately predict phencyclidine disposition in these species. A primary goal of this project was to evaluate the effects of the administration of an anti-phencyclidine monoclonal antibody on phencyclidine disposition and toxicity in the rat. The monoclonal antibody was produced in murine ascites fluid. The antibody was purified using a recirculating isoelectric focusing apparatus. This method provided a rapid technique which can be used to purify monoclonal antibody from large quantities of ascites fluid, yielding reasonably good antibody recovery and very high purity. Characterization of the antibody showed only moderate affinity and high cross reactivity. Administration of the monoclonal antibody did not significantly alter either phencyclidine disposition or toxicity. While qualitative differences in recovery from phencyclidine-induced toxicity occurred in rats receiving the anti-phencyclidine antibody, these differences failed to be statistically different from control rats. These results may be explained by the poor qualities (moderate affinity, high cross-reactivity) of the monoclonal antibody.