Abstract
Here we describe methods for screening human blood to isolate peripheral blood mononuclear cells (PBMCs) capable of binding fluorescently labeled antigen, as well as methods for the amplification and sequencing of B cell receptor (BCR) heavy and light chain genes. Detailed protocols are provided for transient mammalian expression in a hexahistidine-tagged Fab format, purification by immobilized metal affinity chromatography (IMAC), and affinity determination by BioLayer interferometry (BLI).
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Acknowledgments
Protocols for single-cell sequencing BCR heavy and light chain genes are based on methods originally developed by Smith et al. (Nat Protocols, 2009) and were modified in our laboratory at the Garvan Institute. This work was funded by the Garvan Institute of Medical Research, the Australian National Health and Medical Council, the Australian Research Council, and a NSW Health Career Fellowship to Joanne Reed.
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Schofield, P. et al. (2018). Sequencing and Affinity Determination of Antigen-Specific B Lymphocytes from Peripheral Blood. In: Nevoltris, D., Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 1827. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8648-4_15
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DOI: https://doi.org/10.1007/978-1-4939-8648-4_15
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