title: 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.
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.
authors.affiliation: Department of Antibody Engineering, Genentech Inc., South San Francisco, California, USA.
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Department of Antibody Engineering, Genentech Inc., South San Francisco, California, USA.
authors.forename: Nancy Y
authors.initials: NY
authors.lastname: Chiang
authors.affiliation: Department of Infectious Diseases, Genentech Inc., South San Francisco, California, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Chai
authors.affiliation: Department of Antibody Engineering, Genentech Inc., South San Francisco, California, USA.
authors.forename: Dhaya
authors.initials: D
authors.lastname: Seshasayee
authors.affiliation: Department of Translational Immunology, Genentech Inc., South San Francisco, California, USA.
authors.forename: Wyne P
authors.initials: WP
authors.lastname: Lee
authors.affiliation: 1] Department of Translational Immunology, Genentech Inc., South San Francisco, California, USA. [2].
authors.forename: Mercedesz
authors.initials: M
authors.lastname: Balazs
authors.affiliation: Department of Antibody Engineering, Genentech Inc., South San Francisco, California, USA.
authors.forename: Gerald
authors.initials: G
authors.lastname: Nakamura
authors.affiliation: Department of Infectious Diseases, Genentech Inc., South San Francisco, California, USA.
authors.forename: Lee R
authors.initials: LR
authors.lastname: Swem
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000914:Antibodies, Viral / Q000378:metabolism; D000941:Antigens / Q000737:chemistry / Q000378:metabolism*; D001402:B-Lymphocytes / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D009980:Influenza A virus / Q000276:immunology; D016513:Mice, SCID
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000941:Antigens; D007074:Immunoglobulin G