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Schmidthals, Katrin (2013): Generation and characterization of heavy chain antibodies derived from Camelids. Dissertation, LMU München: Fakultät für Biologie



Antibodies and antibody fragments are essential tools in basic research, diagnostics and therapy. Conventional antibodies consist of two heavy and two light chains with both chains contributing to the antigen-binding site. In addition to these conventional antibodies, camelids (llamas, alpacas, dromedaries and camels) possess so-called heavy chain antibodies (hcAbs) that lack the light chains. The antigen binding site of these unusual antibodies is formed by one single domain only, the so called VHH domain. The VHH domain represents the smallest intact antigen binding fragment (~ 15 kDa) and is characterized by very high stability, solubility and specificity. These unique features render VHHs a promising alternative to conventional antibodies and antibody fragments with a multitude of possible applications. In the course of this thesis, we aimed to generate and characterize VHHs suitable for different applications ranging from biochemical studies to immunofluorescence assays and live cell imaging. In order to meet the diverse requirements of the intended downstream applications, a new selection method differing from traditional phage display, called native panning, was developed and established. In combination with the protein-protein interaction fluorescent two-hybrid (F2H) assay, a new process to identify antigen specific VHHs functional inside living cells was developed. We used the immune system of alpacas to generate VHH libraries against various antigens, ranging from small peptides to large proteins. The libraries were screened by phage display and antigen specific VHHs were identified by phage ELISA. We were able to identify antigen specific VHHs against different antigens which were characterized and their functionality was tested in various applications. Furthermore, we could demonstrate that the selection method influences which VHHs are identified and therefore needs to be chosen very carefully with regard to the intended biochemical and cell biological application. In summary, we developed and established an efficient and versatile process to screen and identify antigen specific VHHs suitable for different downstream applications.