Abstract
The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody–secreting plasmablasts can be enriched in vivo, in a severe combined immunodeficient (SCID)/beige mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti–influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.
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Acknowledgements
We are grateful for the flow cytometry expertise of J. Cupp, L. Gilmour, J. Borneo, C.K. Poon and T. Ho. We also thank E. Brown and F. Martin for insightful discussion. We thank Y. Yang, A. Wong, K. Billeci, S. Heldens, P. Hass and K. Schroeder for their technical expertise.
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Z.L., N.Y.C., N.C., W.P.L., M.B. and G.N. performed the experiments; L.R.S., M.B., N.C., G.N. and N.Y.C. developed the methods; and Z.L., G.N., D.S. and L.R.S. wrote the manuscript.
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At the time of this work, Z.L., N.Y.C., N.C., W.P.L., M.B., G.N. and L.R.S. were employees of Genentech, a member of the Roche Group, and may have an equity interest in Roche.
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Lin, Z., Chiang, N., Chai, N. et al. In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells. Nat Protoc 9, 1563–1577 (2014). https://doi.org/10.1038/nprot.2014.104
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DOI: https://doi.org/10.1038/nprot.2014.104
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