Strategies for the detection of endocrine disrupting compounds utilising recombinant oestrogen receptor ligand-binding domains and high specificity monoclonal antibodies
| dc.contributor.advisor | Swart, Pieter | en_ZA |
| dc.contributor.advisor | Swart, Amanda C. | en_ZA |
| dc.contributor.author | Tait, Timo | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Biochemistry. | en_ZA |
| dc.date.accessioned | 2018-11-23T09:56:21Z | |
| dc.date.accessioned | 2018-12-10T06:36:34Z | |
| dc.date.available | 2018-11-23T09:56:21Z | |
| dc.date.available | 2018-12-10T06:36:34Z | |
| dc.date.issued | 2018-12 | |
| dc.description | Thesis (PhD)--Stellenbosch University, 2018. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: During the last two and a half decades, a large body of research has been amassed which indicate that various compounds capable of inducing hormone-like responses in humans and animals are present within the environment. These natural and synthetic molecules, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand-binding domains of these proteins. The current consensus is that endocrine disruptors pose a significant, long-term environmental risk to the well-being of both humans and wildlife. Resultantly, there is a growing need for the development of technologies that can be employed in the rapid assessment of environmental samples for the presence of detrimental xenobiotics. In the view of this need, this dissertation describes membrane-based biological devices which may ultimately form the basis of novel assays which may be used for the rapid detection of endocrine disrupting compounds in remote geographical locations. Specifically, this study describes: 1. A review of the development of in vitro membrane-based immunoassays used in rapid diagnostics, point-of-care analysis and over-the-counter devices for the detection of diseases, environmental contaminants, drugs of abuse and adulterants in food and water supplies. 2. The heterologous expression and purification of highly functional recombinant forms of the alpha isoform of the human oestrogen receptor ligand-binding domain. 3. The characterisation of the ligand-binding properties of these receptor molecules regarding the natural ligand, 17β-oestradiol, other hormones and several known endocrine disruptors by saturation and competitive radioligand-binding methods. 4. Immobilisation of the receptor proteins onto a synthetic support via chelation chemistry and the determination of endocrine active compound sequestration to the biofunctionalised membrane. 5. The establishment, selection and propagation of immortal murine hybridoma cell strains derived from antigen-stimulated B-cells fused to Sp2/0-Ag14 murine myeloma cells. 6. The purification of monoclonal antibodies raised against a defined epitope on the human oestrogen receptor alpha ligand-binding domain surface. 7. Covalent labelling of purified monoclonal antibodies with colloidal gold nanoparticles in preparation for the development of a novel immunochromatographic device. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Die teenwoordigheid van natuurlike en sintetiese chemiese middels wat oor die vermoë beskik om die aksies van hormone in die mens en dier na te boots was ’n belangrike focus in navorsing gedurende die laaste twee en ’n halwe dekades. ’n Verskeidenheid van ontwikkelings-, reproduktiewe- en fisiologiese abnormaliteite ontstaan as gevolg van die aksies van hierdie molekule, genaamd endokriene-ontwrigters, op die natuurlike funksionering van die endokriene-sisteem. Gegewe dat die groep chemiese middels waaruit endokriene-ontwrigters bestaan ’n diverse oorsprong het lei dit daartoe dat algemene analitiese tegnieke nie altyd geskik is vir effektiewe omgewingsmonitering is nie. Die modulasie van geentranskripsie is een van die metodes wat voorgestel word as ’n metode waarop hierdie eksogene molekule die homeostatiese regulering deur die endokriene-sisteem ontwrig. ’n Algemene wyse waarop vele endokrien-ontwrigtende stowwe geentranskripsie beïnvloed is deur interaksie met die hormoon-bindings loki van ’n belangrike groep transkripsiefaktore, die nukluêre reseptore. Die huidige konsensus is dat endokriene ontwrigters ’n beduidende, langtermyn omgewingsrisiko vir die welsyn van mense, sowel as diere inhou. Gevolglik is daar 'n toenemende behoefte aan die ontwikkeling van tegnologieë vir die vinnige nasporing van nadelige xenobiotika in omgewingsmonsters. In die lig van dié behoefte beskryf hierdie proefskrif membraangebaseerde biologiese toestelle wat uiteindelik die basis kan vorm van nuwe toetse vir die spoedige opsporing van endokriene ontwrigtende verbindings in afgeleë geografiese areas. Meer spesifiek beskryf hierdie studie: 1. 'n Oorsig oor die ontwikkeling van in vitro membraan-gebaseerde immunoessaïs wat gebruik word in vinnige diagnostiese analise, punt-van-sorg-analise en oor-die-toonbank-toestelle vir die deteksie van siektes, omgewingsbesoedeling, dwelms en nadelige bymiddels in kos- en waterbronne. 2. Die heteroloë uitdrukking en suiwering van hoogs funksionele rekombinante vorme van die alfaisoform van die menslike estrogeenreseptor ligandbindende domein. 3. Die karakterisering van die ligandbindingseienskappe van hierdie reseptorproteïene ten opsigte van die natuurlike ligand, 17β-estradiol, ander hormone en verskeie bekende endokriene ontwrigters deur versadiging en mededingende radioligand-bindingsmetodes. 4. Immobilisering van die reseptorproteïene op 'n sintetiese membraan deur middel van kelering, gevolg deur toetsing van binding aan die biofunksionele membraan deur verbindings met bekende endokriene aktiwiteite. 5. Die vestiging, seleksie en vermeerdering van hibried muissellyne wat gevorm is deur die versmelting van antigeengestimuleerde B-selle met die Sp2/0-Ag14 muis miëlomasellyn. 6. Die suiwering van monoklonale teenliggaampies wat vervaardig is om 'n gedefinieerde epitoop op die alfa-isovorm van die menslike estrogeenreseptor se ligandbindingsdomein te herken. 7. Kovalente binding van gesuiwerde monoklonale teenliggaampies aan kolloïdale goudnanopartikels in voorbereiding vir die ontwikkeling van 'n nuwe immunochromatografiese toestel. | af_ZA |
| dc.description.version | Doctoral | en_ZA |
| dc.format.extent | xxiii, 200 leaves : illustrations (some color) | |
| dc.identifier.uri | http://hdl.handle.net/10019.1/105204 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Endocrine disrupting chemicals | en_ZA |
| dc.subject | Monoclonal antibodies | en_ZA |
| dc.subject | Estrogen receptor modulators | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.subject | Ligand binding (Biochemistry) | en_ZA |
| dc.title | Strategies for the detection of endocrine disrupting compounds utilising recombinant oestrogen receptor ligand-binding domains and high specificity monoclonal antibodies | en_ZA |
| dc.type | Thesis | en_ZA |