title: Induction of antigen-specific isotype switching by in vitro immunization of human naive B lymphocytes.
abstract: The use of in vitro immunization technology for the generation of human antigen-specific antibodies has essentially resulted in low affinity IgM antibodies, resembling an in vivo primary immune response. We now describe a detailed reproducible protocol for a two-step in vitro immunization, which yields isotype switched, antigen-specific human antibodies. The immunizing antigen was a 30aa synthetic peptide, containing both a B (15aa V3 peptide of the HIV-1) and a T helper cell epitope (15aa peptide from tetanus toxin). The immunization protocol includes: (i) a selection procedure of donors with a memory T cell response against tetanus toxoid; (ii) immunization of mature naive peripheral B lymphocytes in two distinct phases, involving a primary and a secondary step. None of the donors which were examined after primary immunization showed at any time an IgG anti-V3 specific antibody response, while all the donors showed an IgM response. After the secondary immunization step, anti-V3 antibodies of both IgM and IgG isotypes were detected. The switch frequency event was high among the tested donors (5/8).
authors.affiliation: Laboratory of Applied Biochemistry and Immunology, Institute of Molecular Biology and Biotechnology, Heraklion, Greece.
authors.forename: A
authors.initials: A
authors.lastname: Zafiropoulos
authors.forename: E
authors.initials: E
authors.lastname: Andersson
authors.forename: E
authors.initials: E
authors.lastname: Krambovitis
authors.forename: C A
authors.initials: CA
authors.lastname: Borrebaeck
mesh_terms: D000595:Amino Acid Sequence; D000941:Antigens / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D019013:CD40 Antigens / Q000502:physiology; D006801:Humans; D007114:Immunization; D017578:Immunoglobulin Class Switching; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D013745:Tetanus Toxoid / Q000276:immunology
chemical_list: D000941:Antigens; D019013:CD40 Antigens; D013745:Tetanus Toxoid