Establishment of a systematised approach for the development of novel diagnostics and therapeutics in the field of biotechnology

The aim of the first part of this thesis was to generate an antibody against Nonylphenol for later use in biofilters for the elimination of Nonylphenol from waste water. For this purpose mice were immunised following different strategies. The immunisation according to High Zone Tolerisation led to the best results regarding a nonylphenol specific antibody titre in the mouse serum. Successful fusion of the murine B-cells with myeloma cells were performed followed by single cell sorting (FACS). Monoclonal clones could be established and at least 4 stable cell lines showed specific binding to Nonylphenol in ELISA. One antibody was further tested in a ligand fishing approach with subsequent GCMS analysis of the supernatant which proved the binding to NP. The aim of the second part was the establishment of a procedure for the generation of antibodies specific for proteins of lung cancer and pancreatic cancer, respectively, and subsequent identification of these proteins. The approach was designed to prepare the ground for the establishment of a high throughput setup for the proteome wide production of monoclonal antibodies, focusing on the discovery of a compilation of targets with potential diagnostic or therapeutic value. Mice were immunised with whole cells, cell lysates as well as tissue lysates. The development of a specific antibody titre could be proven by ELISA for all antigens. Mouse spleen cells were fused with Sp2/mIL-6 myeloma cells. In the scope of this project the single cell sorting of hybridoma cells could be established followed by the successful culturing of single cells resulting in growth rate of up to 40%. By this 394 and 340 (F. Kampmeier) antibody producing clones were obtained. Only a part of these clones could be analysed due to time and manpower limitations. For antibody production a protein free medium was used. In combination with affinity matrix beads the need of time and money consuming purification steps is circumvented. The antibodies were used for immunoprecipitation experiments and subsequent identification of the antigens by mass spectrometry. The functionality of this approach could be proven by model systems using antibodies of known specificity with lysate spiked with the appropriate antigen. Five new antigens could be identified by this method. The presented results confirm the suitability of the approach for the production and characterisation of antibodies against a variety of targets.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2006

Author dc:creator

• Schumacher, Petra

Contributors dc:contributor

• Fischer, Rainer

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-122779%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61096