Charakterisierung immuninhibitorischer Eigenschaften von SIGIRR und die Herstellung monoklonaler Antikörper gegen humanes SIGIRR

Im ersten Teil der vorliegenden Arbeit wurden immuninhibitorische Eigenschaften SIGIRRs in der Stimulation primärer muriner Zellen untersucht. Der zweite Teil dieser Arbeit beschäftigte sich mit der Herstellung monoklonaler Antikörper gegen humanes SIGIRR. Fragestellung der Arbeit war es, eine Betei...

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Bibliographic Details
Main Author: Kumler, Jasmin Ute
Contributors: Bauer, Stefan (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2016
Online Access:PDF Full Text
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The first part of this thesis focuses on the immuninhitory properties of SIGIRR in the stimulation of primary murine cells. The second part describes the production of monoclonal antibodies against human SIGIRR. The central question of this theses was to distinguish the role of SIGIRR regarding the immunregulation of murine spleen cells, B-cells and myeloid Dendritic Cells via toll-like receptors and the circumstances under which those would come to pass. Monoclonal antibodies against human SIGIRR were to be raised, characterised and used in immunfluorescence-aided expression studies on SIGIRR. For this purpose, for one, different stimuli were used on wildtype versus SIGIRR-knockout mouce cells comparing the resulting IL-6 reaction. Secondly, SIGIRR-knockout mice were inoculated with SIGIRR and hybridoma cells producing SIGIRR-specific antibodies were generated. Antibodies were then tested for usage in ELISA, Western Blot and FACS. In violation of the original hypotheses, the stimulation experiments with different TLG-ligands showed not differences in the IL-6 production between wildtype or SIGIRR-knockout in spleen cells, B-cells or myeloid Dendritic Cells. Hence no immunregulative influence of SIGIRR on TLRsignaling was evident in this experimental environment, though most current studies would suggest otherwise. Solely preliminary tests with MACS-separated CD11c+ mDCs were in favour of the stated theses. The raising of antibodies resulted in SIGIRR-specific antibodies with good binding-properties in ELISA and Western blot. FACS results showed SIGIRR on the surface of HEK cells transfected with SIGIRR and signalpeptide. The results obtained in the stimulation experiments deviated remarkably from the stated thesis and current publications. Therefore the reflection of probable causes as discussed here might be essential for future endeavour. Though some results could be achieved from SIGIRR-specific antibodies, raising SIGIRR-specific antibodies was limited by the ability of hybridoma cells to produce antibodies in continous culture and stable hybridoma cell clones could not be obtained. In future stimulation experiments with SIGIRR-knockout mice the use of MACS-separated CD11c positive myeloid Dendritic Cells seems to be most promising. The raising of SIGIRRspecific antibodies in the laboratory is a time-consuming but promising method, if early subcloning is respected, but purchasing antibodies on the market should also be taken in consideration. SIGIRR-specific antibodies could be used e.g. via fluorescence-marking in studies on the expressionpattern of SIGIRR in different cell lines.