INDUCTION OF ANTIGEN-SPECIFIC ISOTYPE SWITCHING BY IN-VITRO IMMUNIZATION OF HUMAN NAIVE B-LYMPHOCYTES

The use of in vitro immunization technology for the generation of huma n 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 imm unization, which yields isotype switched, antigen-specific human antib odies. 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 agai nst tetanus toroid; (ii) immunization of mature naive peripheral B lym phocytes in two distinct phases, involving a primary and a secondary s tep. None of the donors which were examined after primary immunization showed at any time an IgG anti-V3 specific antibody response, while a ll 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).