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Isolation of human monoclonal antibodies from peripheral blood B cells
Open accessAbstract
Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity.
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Corti, D., Voss, J., Gamblin, S. J., Codoni, G., Macagno, A., Jarrossay, D., Vachieri, S. G., Pinna, D., Minola, A., Vanzetta, F., et al. (2011) · Science · doi:10.1126/science.1205669 · Cited by 1,203
Traggiai, E., Becker, S., Subbarao, K., Kolesnikova, L., Uematsu, Y., Gismondo, M. R., Murphy, B. R., Rappuoli, R., & Lanzavecchia, A. (2004) · Nature Medicine · doi:10.1038/nm1080 · Cited by 697
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Doria-Rose, N. A., Schramm, C. A., Gorman, J., Moore, P. L., Bhiman, J. N., DeKosky, B. J., Ernandes, M. J., Georgiev, I. S., Kim, H. J., Pancera, M., et al. (2014) · Nature · doi:10.1038/nature13036 · Cited by 709
Self-citationReverse vaccinology 2.0: Human immunology instructs vaccine antigen design
Rappuoli, R., Bottomley, M. J., D’Oro, U., Finco, O., & De Gregorio, E. (2016) · Journal of Experimental Medicine · doi:10.1084/jem.20151960 · Cited by 435
Identification of a CD4-Binding-Site Antibody to HIV that Evolved Near-Pan Neutralization Breadth
Huang, J., Kang, B. H., Ishida, E., Zhou, T., Griesman, T., Sheng, Z., Wu, F., Doria-Rose, N. A., Zhang, B., McKee, K., et al. (2016) · Immunity · doi:10.1016/j.immuni.2016.10.027 · Cited by 369
Self-citationKisalu, N. K., Idris, A. H., Weidle, C., Flores-Garcia, Y., Flynn, B. J., Sack, B. K., Murphy, S., Schön, A., Freire, E., Francica, J. R., et al. (2018) · Nature Medicine · doi:10.1038/nm.4512 · Cited by 293
Salem, G. M., Galula, J. U., Wu, S.-R., Liu, J.-H., Chen, Y.-H., Wang, W.-H., Wang, S.-F., Song, C.-S., Chen, F.-C., Abarientos, A. B., et al. (2024) · Communications Biology · doi:10.1038/s42003-023-05661-w · Cited by 14
Metadata
- Authors
- Huang, J., Doria-Rose, N. A., Longo, N. S., Laub, L., Lin, C.-L., Turk, E., Kang, B. H., Migueles, S. A., Bailer, R. T., Mascola, J. R., & Connors, M.
- Venue
- Nature Protocols
- Published
- 2013-09-12
- Volume
- 8
- Issue
- 10
- Pages
- 1907-1915
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W1989544708
- Cited by
- 168
- References
- 2
- FWCI
- 6.89
- Type
- journal article
- Language
- en
- Open access
- green
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- Huang, J., Doria-Rose, N. A., Longo, N. S., Laub, L., Lin, C.-L., Turk, E., Kang, B. H., Migueles, S. A., Bailer, R. T., Mascola, J. R., & Connors, M. (2013). Isolation of human monoclonal antibodies from peripheral blood B cells. Nature Protocols. https://doi.org/10.1038/nprot.2013.117
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