HybriFree: A Robust and Rapid Method for the Development of High Quality Monoclonal Antibodies from Different Host Species

摘要

The production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields. Many different techniques have been described to isolate the antigen-binding domain sequences of antibodies possessing the desired properties. However, most of these techniques have shortcomings. We have developed 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. HybriFree technology can be used to isolate monoclonal antibody sequences from mice, rabbits and chickens. After immunization the crude spleen samples are used for capturing B-cells expressing antigen specific antibodies. cDNA of antibody variable domains is amplified from the captured cells and used as source material for simple and rapid restriction/ligation free cloning. Construction of VH-VL combinatory library can be done either in scFv-Fc or IgG expression plasmid format and directly used for screening purposes as well as for the subsequent production of the developed monoclonal antibodies in mammalian cell culture. The isolated antibodies are functional in different immunoassays, including ELISA, immunofluorescence and Western blot. Also, a modified method including a negative selection step can be used to isolate specific antibodies targeting the desired epitope and eliminate antibodies directed to undesired off-targets. HybriFree technology is a robust and rapid method for 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.