Natural killer (NK)-like lymphocytes have recently been identified in thymectomised (Tx) Xenopus which are capable of spontaneous cytotoxicity towards the MHC- deficient, allogeneic thymus tumour cell line B(_3)B(_7). This Thesis describes attempts to raise antibodies to Xenopus NK cell surface antigens by phage display and hybridoma technology. The phage display technique was optimised for raising antibodies to novel, cellular antigens in a trial run using the Xenopus thymus tumour cell line B(_3)B(_7). Having isolated a phage antibody which was shown by flow cytometry to bind B(_3)B(_7) cells, the technique was then used to try and raise antibodies to Xenopus NK cells. Isolation of an NIC-specific phage antibody was not achieved but phage antibody XL-6 was raised, which bound an antigen on Xenopus lymphocytes. Phage antibody XL-6, and soluble scFv derived from this, were able to identify a putative mature T cell population in the thymus and may be specific for an amphibian homologue of the mammalian leukocyte common antigen CD45. Hybridoma technology was used to isolate three monoclonal antibodies, 1F8, 4D4 and 1G5, which were shown by flow cytometric analysis to identify a putative NK cell population in control and Tx Xenopus. Following immunomagnetic purification, 1F8- positive spleen cells from control and Tx animals were shown to kill the MHC- deficient tumour target B(_3)B(_7), confirming that this antibody was specific for Xenopus NK cells. Western blotting experiments showed that 1F8, 4D4 and 1G5 identified a doublet of protein bands at 72 and 74 kilodaltons in Xenopus gut lymphoid lysates. Initial attempts to isolate cDNA encoding a Xenopus NK cell surface antigen through immunoscreening a xenopus gut cDNA expression library with antibody 1G5 were unsuccessful as was an attempt to clone a Xenopus homologue of the mammalian NK receptor NKR-Pl by PGR.

The aim of this master‘s thesis is to prepare a monoclonal antibody against cyclin-dependent kinase 13 (CDK13). The theoretical part focuses on antigen-antibody binding, which is essential for the use of monoclonal antibodies in the determination of CDK13 as well as the transcription that this kinase affects. This section is also devoted to Western blot and ELISA methods for detection of newly generated antibodies. Furthermore, the antibodies and the antigen definition are stated, which are later on discussed. The practical part is devoted to the preparation of antigen - its isolation and purification on a peristaltic pump. It also addresses immunization, its course, and the amount of antigen used to immunize mice. After immunization, the work focuses on fusion of sp-2 cells and splenocytes, which were first removed from the immunized mouse and purified. After the fusion alone, selection of hybridomas on HAT selection medium is mentioned, followed by detection first by ELISA and later by Western blotting. The resulting hybridomas with positive ELISA response are frozen for further testing at the Veterinary Research Institute in Brno, where the entire practical part of this thesis was carried out. These frozen hybridomas are further tested by immunoprecipitation to conclude this thesis.

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