Abstract
BackgroundThe production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields. A critical step in this process is the isolation of the antigen-binding domain sequences of antibodies possessing the desired properties. Many different techniques have been described to achieve this goal, but all have shortcomings; most techniques have problems with robustness, are time-consuming and costly, or have complications in the transfer from isolation to production phase. Here, we report a novel HybriFree technology for the development of monoclonal antibodies from different species that is robust, rapid, inexpensive and flexible and can be used for the subsequent production of antibodies in mammalian cell factories.ResultsHybriFree technology is illustrated herein via detailed examples of isolating mouse, rabbit and chicken monoclonal antibody sequences from immunized animals. Starting from crude spleen samples, antigen capturing of specific B-cells is performed initially. cDNA of antibody variable domains is amplified from the captured cells and used a source material for simple and rapid restriction/ligation free cloning of expression vector library in order to produce scFv-Fc or intact IgG antibodies. The vectors can be directly used for screening purposes as well as for the subsequent production of the developed monoclonal antibodies in mammalian cell culture. The antibodies isolated by the method have been shown to be functional in different immunoassays, including ELISA, immunofluorescence and Western blot. In addition, we demonstrate that by using a modified method including a negative selection step, we can isolate specific antibodies targeting the desired epitope and eliminate antibodies directed to undesired off-targets.ConclusionsHybriFree can be used for the reliable development of monoclonal antibodies and their subsequent production in mammalian cells. This simple protocol requires neither the culturing of B-cells nor single-cell manipulations, and only standard molecular biology laboratory equipment is needed. In principle, the method is applicable to any species for which antibody cDNA sequence information is available.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0232-6) contains supplementary material, which is available to authorized users.
Highlights
The production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields
The technique is historically referred to as “lymphocyte panning”, and the capture is performed via interactions between the surface immunoglobulin molecules of the B-cells [25]
We found interesting Monoclonal antibodies (MAbs) against human BDNF using the screening of IgG molecules with rabbit VH and VL domains fused with human IgG constant regions
Summary
The production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields. Kivi et al BMC Biotechnology (2016) 16:2 product), the cDNA encoding the variable domains of the heavy (VH) and light chain (VL) must be isolated from the hybridoma cells This step is required for the recombinant production of the final MAb product, as well as for improvements such as humanization, isotype conversion, and affinity maturation. Combinatorial strategies must involve a screening step for the identification of antibodies (VH and VL combinations) with the desired properties from large libraries These screening methods involve in vitro antibody display techniques including phage display [2, 3], ribosome display [4], and in vivo display platforms such as bacterial, yeast, and mammalian cell-surface displays [5]. Instead, performing antibody selection in mammalian cells ensures that the selected antibody is synthesized, modified, assembled and secreted through the same cellular pathways employed in the production phase
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
