Browsing by Person "Hilss, Karen"

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Herstellung monoklonaler Antikörper gegen thermostabile Antigene von Bacillus anthracis zur Anwendung in der Anthraxdiagnostik
(2012) Hilss, Karen; Beyer, Wolfgang
The Ascoli test is a fast and inexpensive diagnostic tool, using polyclonal serum against thermostable antigens of B. anthracis. However, this test is not highly specific for B. anthracis, since the thermostable antigens, on which this test is based, are also present in other Bacillus species and therefore lead to cross-reactivity. By employing monoclonal antibodies against B. anthracis specific thermostable antigens, the cross-reactivity with other Bacillus species could be eliminated. The aim of this study was to generate monoclonal antibodies, which react specifically with the potential B. anthracis specific thermostable antigens. At the beginning thermostable antigen preparations from vegetative B. cereus and B. anthracis cultures, as well as from B. cereus and B. anthracis spores were prepared. These eight antigen preparations were used to immunise rabbits. The resulting polyclonal antisera were used to determine cross-reactivity between B. cereus and B. anthracis in Western Blot analysis. In these cross-reactivity tests two proteins with a molecular weight of approximately 30 and 50 kDa respectively, which are specifically present in antigen preparations of B. anthracis were identified. These proteins do not react with sera of rabbits, immunised with the B. cereus preparations. The 30 kDa protein is present in vegetative and spore preparations of B. anthracis, while the 50 kDa protein is only present in vegetative antigen preparations of B. anthracis. These potentially B. anthracis specific proteins in the vegetative antigen preparation of B. anthracis were partially purified with anion exchange chromatography using FPLC and were used to immunise three BALB/c mice. The spleen of the mouse with the highest specific antibody response was then used to fuse the B-cells with murine myeloma cells in order to generate hybridomas. The supernatants of the resulting hybridomas were screened to identify clones producing antibodies against the thermostable antigens of B. anthracis. After 14 screens the positive clones were divided into two different cell lines. The clones of the V- (vegetative) line were further tested for production of antibodies against the thermostable antigens of vegetative cells of B. anthracis. The S- (spores) line was screened for clones producing antibodies against B. anthracis spore preparations. After two and four screens, the three monoclonal cell lines BaV5, BaV15 and BaV16 were established. The determination of the immunoglobulin class revealed, that BaV5 is a mixed culture with several different antibodies. The cell lines BaV15 and BaV16 produce an immunoglobulin of class M. In determining the specificity of the monoclonal antibodies BaV15 and BaV16 purified from Squarix Biotechnology, no cross-reactivity with 20 vegetative and 10 spore preparations of different Bacillus ssp. (non-anthracis) was found. The purified antibodies, which specifically detect vegetative cells of B. anthracis, were found to be unstable. Trying to stabilize the antibody by additives led to no success, so that further analyses for characterization of the antibodies were not possible.