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Studies on the production, characterisation and applications of monoclonal antibodies and antibody-based conjugates

Cahill, Dolores J. (1994) Studies on the production, characterisation and applications of monoclonal antibodies and antibody-based conjugates. PhD thesis, Dublin City University.

Abstract
The work described in this thesis involved the production of antibodies to several defined antigens and the chemical synthesis of bispecific antibodies. Monoclonal antibodies against glial fibrillary acidic protein (anti-GFAP), and against a GFAP-like protein on the human anaplastic astrocytoma cell line, G-CCM (anti-G-CCM) were produced by electrofusion and by fusion using polyethylene glycol, respectively. These antibodies were purified with saturated ammonium sulphate (S AS) and protein A. These antibodies were fully characterised by ELISA, Isotyping, SDS-PAGE, Immunocylochemisiry, Dot Blot, Western immunoblotting, BIAcore, HPLC, Immunofluoresence and FACS An attempt was made to clone and express the variable regions of the anti-G-CCM monoclonal antibody in Escherichia colt. Iodinated Bolton-Hunter reagent (IBHR) was conjugated to bovine serum albumin (BSA), ovalbumin and keyhole limpet haemocyanin (KLH). These IBHR-protein conjugates were characterised by the iodide assay and the Bradford protein assay. The IBHR-BSA conjugate was used to immunise six mice and a rabbit to produce anti-IBHR antibodies. A monoclonal anti-IBHR antibody was not obtained. However, a polyclonal anti-IBHR antibody was produced and was purified by SAS, protein A and CNBr-activated sepharose affinity chromatography. This antibody was characterised by ELISA, SDS-PAGE and HPLC Bispecific antibodies were chemically generated using (1) anti-GFAP and ana-IBHR and (u) anti-G-CCM and anti-IBHR Ricin is a highly cytotoxic compound Bispecific antibodies were made using an acquired anti-Ricm. A chain monoclonal antibody together with anti-G-CCM and anti-GFAP. These bispecific antibodies were characterised by ELISA, SDS-PAGE, HPLC and FACS and may be used to target radioactive compounds and toxins to astrocytoma cells, in vivo. A catalytic monoclonal antibody was generated using a transition state analogue-KLH conjugate as an immunogen in six mice. This antibody was precipitated using sodium sulphate and purified by protein G affinity chromatography. This antibody catalysed the hydrolysis of the corresponding carbonate and its rate kinetics were determined.
Metadata
Item Type:Thesis (PhD)
Date of Award:1994
Refereed:No
Supervisor(s):O'Kennedy, Richard
Uncontrolled Keywords:Monoclonal antibodies; glial fibrillary acidic protein; anti-GFAP
Subjects:Biological Sciences > Biotechnology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:18389
Deposited On:24 Jun 2013 10:27 by Celine Campbell . Last Modified 24 Jun 2013 10:27
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