Publikationsserver der RWTH Aachen University
Generierung, Charakterisierung und funktionaler Assay von Antikörpern und Antikörperfragmenten gegen die HCV Strukturproteine core, E1 und E2
Abstract
dc:descriptionThe Hepatitis C virus constitute a global health problem and is the most important member of non-A and non-B Hepatitis C viruses. Hepatitis C is deemed to be one of the most fatal diseases worldwide. Approx. 30% of the 130 million Hepatitis C patients who carry a chronically Infection are developing cirrhosis of their liver or hepatocellular carcinoma. In the dissertation on hand, antibodies against the Hepatitis C (subtype 1b) structure proteins core, E1 and E2 were synthesised. The coding sequences of full-length and deleted versions of the protein mentioned above served as starting material. In a first step sufficient amount of the respective protein was produced in E. coli for the later use as antigen in further analyses and immunisations and purified from the compartment of accumulation e.g. the cytoplasm or periplasm. SDS-PAGE, Immunoblot as well as MS/MS analysis were used to verify the recombinant produced structure proteins. Additionally synthetic peptides were designed as antigens, which represented specific internal amino acid sequences of the native proteins. The peptide synthesis was commercially ordered. In a second step already existing hybridoma clones were screened for their specificity by indirect ELISA. As part of a cooperation project monoclonal antibodies of one of the selected clones were utilised successfully in immunofluorescence analyses. By carrying out a “Two Step Cloning” two recombinant scFv antibodies were generated from the hybridoma originated monoclonal antibodies. These scFv antibodies also showed antigen specificity in ELISA. The corresponding amino acid sequences were analysed with regard to the totality and integrity. In a final step the E. coli produced and verified E2 antigens were mobilised for immunisation of mice resulting in the generation of an additional monoclonal antibody by application of the hybridoma technology. Moreover the recombinant antigens as well as the artificial peptide antigens were used in a second cooperation project for screening of a human Hepatitis phage-library. Arising from these analysis thirteen recombinant antibodies were identified and their amino acid sequences were further characterised.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2008
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Franken, Tobias
- Contributors dc:contributor
-
- Fischer, Rainer
Subjects
dc:subject × 10Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger