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Detection of Edcs Using Recominant Human Eralpha and Monoclonal Strategies for the detection of endocrine disrupting compounds utilising recombinant oestrogen receptor ligand-binding domains and high specificity monoclonal antibodies by Timo Tait Dissertation presented for the degree of Doctor of Philosophy in the Faculty of Science at Stellenbosch University Supervisor: Prof Pieter Swart Co-Supervisor: Prof Amanda Swart December 2018 Stellenbosch University https://scholar.sun.ac.za DECLARATION By submitting this dissertation electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third-party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Timo Tait December 2018 Copyright © 2018 Stellenbosch University All rights reserved Stellenbosch University https://scholar.sun.ac.za When I first entered the study of hormone action, some 25 years ago, there was a widespread feeling among biologists that hormone action could not be studied meaningfully in the absence of organized cell structure. However, as I reflected on the history of biochemistry, it seemed to me there was a real possibility that hormones might act at the molecular level. o Earl W. Sutherland, Nobel Address, 1971 i Stellenbosch University https://scholar.sun.ac.za SUMMARY 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. ii Stellenbosch University https://scholar.sun.ac.za 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 alfa- isoform 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 goud- nanopartikels in voorbereiding vir die ontwikkeling van 'n nuwe immunochromatografiese toestel. iii Stellenbosch University https://scholar.sun.ac.za ACKNOWLEDGEMENTS There are many people who have greatly supported me during the last few years, several of whom have greatly impacted on the success of this study. Foremost, I would like to express my sincerest gratitude to Prof Pieter Swart, my promotor and mentor, for his patience and motivation. His immense knowledge, guidance and continual trust in the directions took during my research helped me in all the facets involved in the completion of a doctoral degree. I could not have imagined having a better advisor and mentor throughout my studies. Besides my advisor, I would like to thank the evaluators of this thesis, especially Prof Marina Rautenbach, for their insightful comments which improved the quality of this document. My sincerest thanks also to Prof Amanda Swart for her keen understanding of scientific writing, her wonderful words of encouragement and providing a broader perspective on the contents of this thesis. To everyone with whom I have had the pleasure of sharing the laboratory during the last few years. You have been like family to me and have helped me grow in tremendous ways. Thank you very much to Ralie Louw for her technical and administrative expertise. Without her valuable support conducting this research would have been an immense challenge. I would especially like to thank my wonderful friends, Jonathan, Therina, Stefan and Louwrens, for all the stimulating discussions, squash, late-nights, early morning coffees, interesting experiments and fun we had during the last few years, both inside and out of the laboratory. There have been many variables along the way, you have been constants. I would also like to thank Prof Edmund Pool from UWC for giving me a crash course in the generation of hybridomas and to Noël Markgraaff and Judith Farao for their invaluable assistance in the animal house. At Stellenbosch University, we are lucky
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