No description available.

No description available.

No description available.

No description available.

by Yeung Shing On. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1989 / Bibliography: leaves 91-112.

While the last century of medical discoveries has made a significant impact on improving the lives of human populations across the globe, a perfect solution to the yearly infection cycle from the influenza virus has yet to be discovered. Although vaccines stand the best chance at targeting yearly epidemics, new treatment options must be created to combat the arrival of rapidly mutating and antiviral-resistant strains of the virus that could lead to another pandemic such as the 1918 Spanish flu that killed millions worldwide. We describe a method to create functionally enhanced monoclonal antibodies targeting influenza via genetic engineering of fragment crystallizable glycan structures. Muscle and liver cell lines were lentivirally-transduced to produce the broadly neutralizing antibody, Fi6v3, while also overexpressing a critical glycosylation enzyme, B-1,4-N-acetyl-glucosaminyltransferase III. Secreted antibodies were tested for effector functionality using a Natural Killer cell degranulation assay and an antibody-dependent cellular phagocytosis assay. Results conclude that modified antibodies from both muscle and liver cells lines exhibit enhanced function in comparison to their unmodified counterparts, providing support to the future creation of an influenza prophylactic or treatment option using antibodies with the ability to more effectively activate innate immune killing mechanisms.

Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies (MAbs), due to their ability to perform correct post-translational modifications. A major issue for use of CHO cells lines for the production of recombinant proteins is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval. Protein production is complex and is influenced by cell growth, transcription, translation, protein folding and post-translational processing and secretory events, which may interact to determine stability of expression during prolonged culture. This thesis aims to identify features associated with stability/instability of recombinant protein expression and methods to improve protein production, with the addition of chemically defined (CD) feed and chemicals. Two exemplar CHO cell lines, which secrete the same recombinant antibody were characterised in response to LTC, feed and DMSO addition. Both cell lines (3.90 and 51.69) exhibited unstable protein production over LTC, with a loss in final antibody titres and specific productivity (Qp). The instability observed within the exemplar cell lines was not due to decreased recombinant gene copy numbers or mRNA expression but was associated with lower viable cell densities, increased ER stress (GADD153 and spliced XBP-1 [XBP-1(s)]) and enhanced rates of lactate utilisation (observed during the decline phase of batch culture). Improvement of recombinant protein expression in response to feed or DMSO addition was associated with lower expression of ER stress markers (ATF4, XBP-1(s) and GADD153 at mRNA level and GADD153 at protein level) and alterations to the metabolic activity of the cultures (prevention of alanine and lactate re-utilisation, and greater glucose utilisation between the stationary and decline phase of batch culture).Although feed or DMSO addition improved recombinant protein production, these additions did not reverse the appearance or progression of instability for cells after LTC. ER stress expression was not abolished as a consequence of feed or DMSO addition. Expression of stress markers at earlier time points may be the factor that limits antibody production and secretion. The consequences of the presence of feed and DMSO addition on ER stress markers and antibody production serves to highlight approaches that may be utilised for engineering more productive or stable protein production phenotypes in parental cell lines.

No description available.

Kwok Leung Wong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 149-170). / Acknowledgments --- p.i / Abstract --- p.ii -v / Abbreviations --- p.vi-vii / Chapter Chapter 1 : --- General Introduction --- p.1 / Chapter Chapter 2 : --- Development of Polyclonal Antibodies Against 6-B-hydroxycortisol （6-B-OHC) And Its Applications / Chapter 2.1 : --- Introduction --- p.40 / Chapter 2.2 : --- Materials and methods --- p.43 / Chapter 2.3: --- Results --- p.55 / Chapter 2.4: --- Discussion --- p.73 / Chapter Chapter 3 : --- Development of Monoclonal Antibody-Based ELISA Against 6-B-hydroxycortisol (6-B-OHC) And Its Applications / Chapter 3.1 : --- Introduction --- p.76 / Chapter 3.2 : --- Materials and methods --- p.89 / Chapter 3.3: --- Results --- p.108 / Chapter 3.4: --- Discussion --- p.135 / Chapter Chapter 4 --- : General Conclusion --- p.141 / References --- p.149

The objective of this study was to produce a monoclonal antibody specific for the microgametocytes of Eimeria tenella, examine the site and stage specificity of the antibody, and investigate the immunopotency of the antibody. BALB/c mice were immunized with antigen containing Eimeria tenella microgametocytes isolated from in vitro systems. After three intraperitoneal immunizations with the antigen and one booster immunization administered by tail vein injection, the mice were sacrificed and their spleen cells fused with SP2/0 mouse myeloma cells using polyethylene glycol as a fusing agent. Resultant hybridomas were screened by immunoelectrophoresis, indirect immunofluorescent antibody assay, and immunoelectron microscopy to determine the isotype, subisotype, site and stages pecificity of the antibody. Of four 96 well plates seeded with fusion products, four hybridomas were found to be producing anti body specific for the target antigen. Only the most strongly positive of these hybridomas, clone T1A3B9, was used for the study. The antibody produced by this hybridoma was found to be of sub isotype IgG2b. T1A389 monoclonal antibody was introduced into Eimeria tenella infected cell cultures on days four, five, and six post-infection. At seven days post-infection, oocyst production was assayed by fixing, staining, and counting the resultant oocysts. Results of the in vitro experiments showed a greater than 50X reduction in oocyst product ion in experimental cultures over controls. Statistical significance of the data were confirmed by a Mann-Whitney U Test. These results indicate that the monoclonal a ntibod y was exert ing an inhibitory effect on the fertilization process. T1A3B9 monoclonal antibody was incubated with Eimeria tenella infected cecal scrapings and cell culture material, immunolabeled with colloidal gold conjugates, and observed by electron microscopy. Results showed that the antibody was binding to the microgametocytes and to no other life cycle stages of the parasite, nor was it binding to host tissue. This indicates that the antibody is stage specific. Additionally, the antibody was seen to bind only to areas in close proximity to the budding flagella of developing microgametes, thus indicating distinct site specificity.