Efficient Cloning Method for Variable Region Genes of Antigen Specific Human Monoclonal Antibody

Abstract

We have developed an in vitro immunization protocol of human peripheral blood mononuclear cells (PBMC) for generating human antigen-specific antibodies. By using this protocol, B cells producing antigen specific antibody can be propagated within a week. In the present study, we tried to establish an efficient strategy to clone variable region genes of antigen specific human monoclonal antibody by applying in vitro immunized PBMC to the phage display method. To evaluate the efficiency of our strategy, heavy and light chain variable region genes were prepared by PCR from PBMC immunized in vitro with mite-extract (ME), and the combinatorial library (>10⁵ members) cloned for display on the surface of a phage. Phage was subjected to a streptavidin magnetic bead panning procedure. After concentrating the MEspecific phage antibody by panning, the phage antibodies specific for ME were detected by ELISA. Results show that rapidly production of antigen-specific human monoclonal antibody from smallish (1.6 × 10⁵) library is possible by using in vitro immunized PBL.