Production of anti-human cannabinoid receptor 2 (CB2) monoclonal antibody using non-viral vectorinduced human CB2 expressing myeloma as an immunogen
Jindaphun Kanyaruck,1 Takheaw Nuchjira,1,2 Putpim Chaochetdhapada,3 Laopajon Witida,1,2 Kotemul Kamonporn,1 Chaiwut Ratthakorn,1 Pata Supansa,1,2 Kasinrerk Watchara1,2
Affiliations:
1 Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
2 Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at the Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
3 Laboratory Animal Center, Chiang Mai University, Chiang Mai, Thailand
Abstract
Background: Cannabinoid receptor 2 (CB2) of the cannabinoid system is predominantly expressed on immune cells and involved in a diverse range of immune functions. However, the role of CB2 in immunoregulation is still controversial. One of the challenges in the detailed characterization and functional study of CB2 is the lack of CB2-specific monoclonal antibodies (mAbs).
Objective: We aimed to produce mAbs against a native form of human CB2 using human CB2 expressing mouse myeloma cells as immunogens.
Methods: Non-viral vector expression system was used to generate stable human CB2-expressing mouse myeloma cells and were utilized as an immunogen for mouse immunization. Hybridoma technique was employed in the production of mAbs. The produced mAbs were verified by flow cytometry and western blotting.
Results: Using a non-viral vector expression system, myeloma clones, which stable expressed human CB2, were generated and used as immunogen for antibody production. Following mouse immunization process, the anti-CB2 polyclonal antibodies were induced. By hybridoma technique, a mAb against CB2 could be generated. This mAb reacted to CB2-expressing THP-1 cells, but not to non-CB2-expressing SH-SY5Y cells. By western blotting, the generated anti-CB2 mAb reacted with a 42 kDa protein presented in lysates of CB2-expressing THP-1 cells, but not with non-CB2-expressing SH-SY5Y cell lysates.
Conclusion: A new approach using human CB2 expressing myeloma cells as immunogen for production of anti-CB2 mAb was developed. The generated anti-human CB2 mAb is regarded as a valuable tool for CB2 characterization. Moreover, the developed technique can be applied to produce other antibodies of interest.
Key words: Cannabinoid system, Cannabinoid receptor, Non-viral vector expression system, Monoclonal antibody production, Anti-cannabinoid receptor 2 antibody