Abstract
DNA immunization with in vivo electroporation is an efficient alternative protocol for the production of monoclonal antibodies (mAb). Generation of mAb by DNA immunization is a novel approach to circumvent the following technical hurdles associated with problematic antigens: low abundance and protein instability and use of recombinant proteins that lack posttranslational modifications. This chapter describes the use of a DNA-based immunization protocol for the production of mAb against a house dust mite allergen, designated as Blo t 11, which is a paramyosin homologue found in Blomia tropicalis mites. The Blo t 11 cDNA fused at the N terminus to the sequence of a signal peptide was cloned into the pCI mammalian expression vector. The DNA construct was injected intramuscularly with in vivo electroporation into mice, and the specific antibody production in mice was analyzed by enzyme-linked immunosorbent assay (ELISA). Hybridomas were generated by fusing mouse splenocytes with myeloma cells using the ClonaCell™-HY Hybridoma Cloning Kit. Six hybridoma clones secreting Blo t 11 mAb were successfully generated, and these mAb are useful reagents for immunoaffinity purification and immunoassays.
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Acknowledgments
This study was supported by grants from the Agency for Science, Technology, and Research (A*STAR), Singapore (R-178-000-010-303, R-178-000-101-305), and Academic Research Fund, National University of Singapore, Singapore (R-178-000-010-112).
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Chua, K.Y., Ramos, J.D., Cheong, N. (2008). Production of Monoclonal Antibody by DNA Immunization with Electroporation. In: Li, S. (eds) Electroporation Protocols. Methods in Molecular Biology™, vol 423. Humana Press. https://doi.org/10.1007/978-1-59745-194-9_40
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DOI: https://doi.org/10.1007/978-1-59745-194-9_40
Publisher Name: Humana Press
Print ISBN: 978-1-58829-877-5
Online ISBN: 978-1-59745-194-9
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