![]() ![]() ![]() 2, middle panel), where different antigen-avidin complexes can be linked to the cell surface and the secreted antibodies can be tested for a specific binding. This approach can be extended to a cross-reactivity screening (Fig. The antigen is specifically recognized by the secreted antibody and the detection takes place via a secondary antibody labelled to a fluorescent dye. 2, left) is performed by an antigen-avidin complex bound to the biotinylated cell. The system allows a combination of three possible sorting options. with the streptavidin-conjugated antigen or isotype-specific antibody, which in turn catches the produced antibody, and a labeled indicator anti-immunoglobulin or antigen (Fig. Such a surface marker, after in vitro biotinylation, should be applicable for the isolation of antigen-specific antibody-producing hybridoma, allowing for a built-up of a bridge e.g. We transfected the myeloma cells to be used for hybridoma fusion with a construct enabling the expression of a surface marker containing the acceptor peptide (AP) sequence for site-specific biotinylation by biotin ligase (BirA) 13. We, therefore, tried to replace this principle by a more gentle method. However, chemical surface labeling is very often unpredictable and may disturb normal functions and the vitality of the cells. To realize this, a covalent surface labeling of antibody-producing cells with biotin was accomplished in the past, which allowed the isolation of specific cells by means of avidin- or streptavidin-conjugated ligands binding the released antibodies 12. To confer this basic principle to the hybridoma technique would require to capture the synthesized antibody on the surface of the synthesizing hybridoma cell (Fig. Such precondition can easily be fulfilled when recombinant antibody fragments are isolated, e.g. To facilitate the isolation of specific antibody-producing hybridomas, a method has to be established which temporarily restricts the cells from releasing the antibody into the culture medium and thus retaining the genotype (the antibody-coding genes) and the phenotype (the produced antibodies) in one entity. Unfortunately, this process could not be combined with a simultaneous, proper validation of the desired antibodies because the concentration in the supernatants are often very low at the early beginning of culture. Therefore, it is necessary to perform limited dilution techniques in order to separate single cells to ensure monoclonality. ![]() The critical issue in the development of antigen-specific hybridomas is the lack of any direct connection between the hybridoma cell and the released antibody. Although several improvements have been tried in the course of the past years, the basic method is still very similar to the original method published by Köhler and Milstein 9, 10, 11. Especially the hybridoma technique which results in full-length monoclonal antibodies can be cumbersome, labour-intensive and time-consuming (Fig. Currently, the reliability of monoclonal antibodies was critically discussed in several publications 4, 5 which is related to a growing demand of better validation and characterization of these molecules 6, 7, 8. Both methods have intrinsic advantages but also difficulties such that they are restricted to specialized laboratories and companies. Most murine antibodies are produced today by means of the hybridoma technique as monoclonal antibodies 2 or with the help of antibody gene libraries and display techniques as recombinant antibody fragments 3. 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.Īntibodies are well known as universal binding molecules with a high specificity for their corresponding antigens and have found, therefore, widespread use in very many different areas of biology and medicine 1. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. 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. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. 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. 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. ![]()
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