Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

the use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. to address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. for the design of the construct we combined the transmembrane domain of the eGf-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. the stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen-and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.

No abstract

No abstract

Photodynamic therapy (PDT) is a mild but effective method to treat certain types of cancer upon irradiation with visible light. Here, three isomeric methylpyridinium alkynylanthracenes 1o─p were evaluated as sensitizers for PDT. Upon irradiation with blue or green light, all three compounds show the ability to initiate strand breaks of plasmid DNA. The mayor species responsible for cleavage is singlet oxygen (1O2) as confirmed by scavenging reagents. Only isomers 1m and 1p can be incorporated into HeLa cells, whereas isomer 1o cannot permeate through the membrane. While isomer 1m targets the cell nucleus, isomer 1p assembles in the cellular cytoplasm and impacts the cellular integrity. This is in accordance with a moderate toxicity of 1p in the dark, whereas 1m exhibits no dark toxicity. Both isomers are suitable as PDT reagents, with a CC50 of 3 μm and 75 nm, for 1p and 1m, respectively. Thus, derivative 1m, which can be easily synthesized, becomes an interesting candidate for cancer therapy.