The goal of this thesis was the development of an easy-to-handle and reliable diagnostic system to discriminate between HCV genotypes 1b, 2 and 3. The envelope protein E2 sequence of genotype 1b was isolated from patients blood whereas consenus sequences had to be deduced from all available genotype-2 and -3 sequences. Sequences were derived from all sequences available in the Los Alamos database. After sequence synthesis, expression was performed in E. coli as well as in different eukaryotic cells. To faciliate protein expression, the C-terminal transmembrane region of E2 genotypes was deleted, leading to a protein shortened by 80 amino acids. By deleting more than the original transmembrane region, the resulting protein amount could be enhanced four-fold. Testing different expression systems in E. coli, most protein was observed using pET26b+ (Novagen). Working with denaturing conditions led to 95% pure protein. Next to prokaryotic expression, different mammalian cells were tested to express the recombinant proteins. Protein purification of positive transfected cells couldnot be shown by purification and western-blotting or by flow cytometry. Besides full-length proteins also specific peptides were deduced from all available E2 genotype-1, -2 and -3 sequences. The peptides were located in a relatively constant region with 85% homology between the different genotypes. To gain monoclonal antibodies against HCV E2, different proteins were used for immunization. Therefore mice were injected with two different E2(1b)-fragments , full-length E2(1b) and the different peptides. By this, 13 genotype-cross-reacting antibodies could be produced. By working with different fragments of E2, the different antibodies recognize various epitopes, making them all eligible candidates for a sandwich ELISA setup. Besides this, the genotype-cross-reacting character and the different binding activities to the three genotype proteins make an exclusion diagnosis possible. To improve antibody affinity to the different genotypes scFv were generated using a new primer set facilitating a two-step cloning strategy. After showing the functionality of the newly established scFv, in vitro evolution was conducted. Therefore random-error prone PCR with additional base analogue hmdCTP in the dNTP mix was used. This lead to a good mutation efficiency and random amino-acid changes could be induced. Later on, phage display technology was used to select new binders. Neither with different E. coli strains nor various panning conditions improved binders could be detected. Until now, an antibody-based detection of HCV genotypes is not possible, but the designed peptides can be used to detect anti-HCV antibodies in direct ELISA. Until now sera of patients with genotype-1 infections were tested and proved the ability of peptides to be used in a diagnostic assay. To demonstrate the efficiancy of this system, more samples of patients with different genotype infections have to be collected and tested with the different genotype peptides.
