published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

No description available.

Isolated in 1994, the <i>BRCA1</i> gene encodes a 1863 amino acid protein, the role of which remains undetermined. <i>BRCA1 </i>germ-line mutations, which result in loss of functional full-length protein, are associated with familial breast and ovarian cancer. This would suggest an important tumour suppressor role for the BRCA1 protein. No somatic mutations have been described in <i>BRCA1</i> in sporadic breast cancer cases. Defective transcription or post transcriptional modification of the BRCA1 protein may however precipitate the same outcome of a loss-of-function protein product facilitating the development of sporadic breast cancer as indeed has already been suggested. Progress in the study of BRCA1 has been impeded by the lack of availability of specific, sensitive antibodies. These problems have led to confusion regarding the sub-cellular localisation and postulated functions of BRCA1. This study has endeavoured to answer three questions. Firstly, are the monoclonal antibodies used specific for the BRCA1 protein? Secondly, by immunohistochemical techniques, what is the sub-cellular localisation of the BRCA1 protein within a cohort of primary sporadic breast cancers? And finally, what is the relationship between BRCA1 expression and pathological, biological and survival parameters within a group of 200 primary sporadic breast cancer cases? Results demonstrate Ab2 to detect a 110 kDa protein consistent with the D11b BRCA1 splice variant. This localised to the cytoplasm in both immunohistochemical and western blot settings. Initial pilot study data suggested Ab2 labelling of sporadic breast cancer cases to be of prognostic significance. This was not supported in an expanded study of 200 cases which confirmed that intensity of immunohistochemical labelling was not an independent prognostic indicator in sporadic breast cancer. Ab1 was confirmed to be non-specific.

No description available.

No description available.

No description available.

No description available.

INTRODUCTION: Since the eradication of smallpox in 1977, mass vaccination efforts against it have been discontinued. Thus, the majority of the younger population is susceptible to both smallpox virus and vaccinia virus (VV). The re-emergence or intentional release of smallpox will present a serious threat to global health. There are limited supplies of smallpox vaccine, which is associated with significant complications, and pooled anti-VV human immune globulin (VIG) that can be used as prophylaxis or to treat smallpox-exposed individuals. We are developing murine monoclonal antibodies (MAbs) able to neutralize VV. The developed MAbs may be useful in establishing a rapid diagnostic test for the detection of VV infection or providing the genetic materials needed for developing recombinant antibodies suitable for human use. METHODS: VV Western Reserve (WR) strain was propagated in HeLa or Chicken Embryo Fibroblast (CEF) cell lines, purified through a 36% sucrose cushion and inactivated by binary ethyleneimine (BEI). Female BABL/c mice were immunized with inactivated VV. Hybridoma cell lines (HCLs) were developed from spleen cells of the mice with high neutralizing antibody titers. Tissue culture supernatants from the developed HCLs were screened by Enzyme-Linked Immunosorbent Assay (ELISA) and Plaque Reduction Assay (PRA) for their abilities to produce neutralizing antibodies against VV. HCLs producing neutralizing antibodies were sub-cloned by limiting dilution method. Highly neutralizing MAbs were isotyped and purified. The effect of using increasing microgram amounts of each MAb or mixtures of two MAbs on VV neutralization has been determined. Specific target proteins recognized by MAbs were detected by western blot assay (WB). The abilities of the developed MAbs to neutralize other three VV strains, Large-variant (L-variant), IHD-W and New York City Board of Health (NYCBH), were measured. RESULTS: We have developed 261 HCLs producing anti-VV antibodies; 65 of them neutralized VV. Twelve HCLs were sub-cloned. We developed 79 sub-clones producing neutralizing MAbs. The majority of them were immunoglobulin IgG1/κ isotype. Four highly neutralizing MAbs were concentrated and purified. They were able to neutralize 50% of VV infection at 0.01-0.1 µg in PRAs. Synergistic effects on VV neutralization were observed when mixing two MAbs from clones, 1-E9-1-E4 and 2-B7-9-E6, at the amounts giving about 20% and 40% VV neutralization. Based on the WB results, the developed MAbs are recognizing 75 kilodalton (kDa), 45 kDa, 35 kDa or 8 kDa WR VV proteins. The abilities of the developed MAbs to neutralize other strains of VV varied. CONCLUSIONS: Several HCLs producing antibodies against VV were developed. Highly neutralizing MAbs against WR VV have been produced and purified. Virus neutralization is dose dependent and some of MAbs have synergistic neutralization effects on each other. Most of the MAbs were targeting the same three virus envelope proteins indicating that these proteins contain important epitope(s) responsible for the neutralizing effects by the developed MAbs. Variable neutralization abilities were observed on three other VV strains indicating their immunobiologic differences with WR VV strain. The developed MAbs may be used as a research tool to study VV pathogenesis or for the development of chimeric antibodies for clinical applications.

INTRODUCTION: Since the eradication of smallpox in 1977, mass vaccination efforts against it have been discontinued. Thus, the majority of the younger population is susceptible to both smallpox virus and vaccinia virus (VV). The re-emergence or intentional release of smallpox will present a serious threat to global health. There are limited supplies of smallpox vaccine, which is associated with significant complications, and pooled anti-VV human immune globulin (VIG) that can be used as prophylaxis or to treat smallpox-exposed individuals. We are developing murine monoclonal antibodies (MAbs) able to neutralize VV. The developed MAbs may be useful in establishing a rapid diagnostic test for the detection of VV infection or providing the genetic materials needed for developing recombinant antibodies suitable for human use. METHODS: VV Western Reserve (WR) strain was propagated in HeLa or Chicken Embryo Fibroblast (CEF) cell lines, purified through a 36% sucrose cushion and inactivated by binary ethyleneimine (BEI). Female BABL/c mice were immunized with inactivated VV. Hybridoma cell lines (HCLs) were developed from spleen cells of the mice with high neutralizing antibody titers. Tissue culture supernatants from the developed HCLs were screened by Enzyme-Linked Immunosorbent Assay (ELISA) and Plaque Reduction Assay (PRA) for their abilities to produce neutralizing antibodies against VV. HCLs producing neutralizing antibodies were sub-cloned by limiting dilution method. Highly neutralizing MAbs were isotyped and purified. The effect of using increasing microgram amounts of each MAb or mixtures of two MAbs on VV neutralization has been determined. Specific target proteins recognized by MAbs were detected by western blot assay (WB). The abilities of the developed MAbs to neutralize other three VV strains, Large-variant (L-variant), IHD-W and New York City Board of Health (NYCBH), were measured. RESULTS: We have developed 261 HCLs producing anti-VV antibodies; 65 of them neutralized VV. Twelve HCLs were sub-cloned. We developed 79 sub-clones producing neutralizing MAbs. The majority of them were immunoglobulin IgG1/κ isotype. Four highly neutralizing MAbs were concentrated and purified. They were able to neutralize 50% of VV infection at 0.01-0.1 µg in PRAs. Synergistic effects on VV neutralization were observed when mixing two MAbs from clones, 1-E9-1-E4 and 2-B7-9-E6, at the amounts giving about 20% and 40% VV neutralization. Based on the WB results, the developed MAbs are recognizing 75 kilodalton (kDa), 45 kDa, 35 kDa or 8 kDa WR VV proteins. The abilities of the developed MAbs to neutralize other strains of VV varied. CONCLUSIONS: Several HCLs producing antibodies against VV were developed. Highly neutralizing MAbs against WR VV have been produced and purified. Virus neutralization is dose dependent and some of MAbs have synergistic neutralization effects on each other. Most of the MAbs were targeting the same three virus envelope proteins indicating that these proteins contain important epitope(s) responsible for the neutralizing effects by the developed MAbs. Variable neutralization abilities were observed on three other VV strains indicating their immunobiologic differences with WR VV strain. The developed MAbs may be used as a research tool to study VV pathogenesis or for the development of chimeric antibodies for clinical applications.