Efficient Acquisition of Antigen-Specific Human Monoclonal Antibody by Using Peripheral Blood Mononuclear Cells Immunized In Vitro

We have developed an in vitro immunization protocol of human ­peripheral blood mononuclear cells (PBMC) for generating human antigen-specific antibodies. Upon sensitization of PBMC with antigen in vitro according to the protocol, B cells producing antigen-specific antibody can be propagated within a week. In the present study, we tried to establish a strategy to clone variable region genes of antigen specific human monoclonal antibody by applying in vitro immunized PBMC to the phage display method. By using PBMC immunized in vitro as template, heavy and light chain variable region genes were easily amplified by PCR. After generating the combinatorial phage library (1.6 × 105 members), phage antibody library was subjected to panning using biotinylated antigen and streptavidin magnetic beads to select antigen-specific phage antibody. After five rounds of panning, we obtained four antigen-specific clones. We combined variable region genes of these selected clones with human IgG constant region genes, and produced as human IgG format antibody. Among these clones, 1C11 showed a highest reactivity for sensitizing antigen. All these results demonstrate that we could obtain antigen-specific human monoclonal antibody from a relatively small phage antibody library by using in vitro immunized PBMC.