title: A rapid and efficient strategy to generate antigen-specific human monoclonal antibody by in vitro immunization and the phage display method.
abstract: An in vitro immunization (IVI) protocol was developed for inducing antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs). After antigen sensitization of PBMCs by IVI, B cells producing antigen-specific antibody can be propagated within a week. Here, we attempted to establish a rapid, efficient strategy to obtain antigen-specific antibody by the phage display method using in vitro immunized PBMCs. Heavy and light chain variable region genes were easily amplified from these PBMCs immunized with mite extract (ME). After generating a combinatorial phage library (1.6 x 10(5) members), 4 antigen-specific clones were selected by 5 panning rounds using biotinylated antigen and streptavidin magnetic beads. Next, we combined variable region genes of these selected clones with human IgG constant region genes and produced human IgG-type antibody. Direct and competitive enzyme-linked immunosorbent assays demonstrated that the mAb 1C11 clone bound specifically to ME. We thus established a rapid, efficient method to obtain antigen-specific human antibody genes and produce human monoclonal IgG antibody using the phage antibody library generated from in vitro immunized PBMCs.
authors.affiliation: Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Shin-ei
authors.initials: SE
authors.lastname: Matsumoto
authors.forename: Makiko
authors.initials: M
authors.lastname: Yamashita
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Katakura
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Aiba
authors.forename: Kosuke
authors.initials: K
authors.lastname: Tomimatsu
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kabayama
authors.forename: Kiichiro
authors.initials: K
authors.lastname: Teruya
authors.forename: Sanetaka
authors.initials: S
authors.lastname: Shirahata
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D039741:Antigens, Dermatophagoides / Q000276:immunology*; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology*; D019151:Peptide Library; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D007074:Immunoglobulin G; D019151:Peptide Library; D011994:Recombinant Proteins