title: Analysis of cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulin.
abstract: Although various in vitro immunization methods to generate antigen-specific antibodies have been described, a highly effective method that can generate high-affinity immunoglobulins has not yet been reported. Herein, we analyzed a cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulins. We identified a combination of T cell-dependent stimuli (IL-4, IL-5, anti-CD38 and anti-CD40 antibodies) plus lipopolysaccharides (LPS) that stimulates antigen-exposed splenocytes in vitro, followed by induction of the cells phenotypically equivalent to germinal center B cells. We also observed that LPS induced high expression levels of mRNA for activation-induced cytidine deaminase. We stimulated antigen-exposed splenocytes, followed by the accumulation of mutations in immunoglobulin genes. From the immunized splenocytes, hybridoma clones secreting antigen-specific immunoglobulins were obtained.
authors.affiliation: Innovative Antibody Engineering Laboratory, Advance Co. Ltd., 5-7 Kobunacho, Chuo-ku, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Inagaki
authors.forename: Tatsunari
authors.initials: T
authors.lastname: Yoshimi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kawahara
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D000915:Antibody Affinity; D000941:Antigens / Q000276:immunology; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D003564:Cytidine Deaminase / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D005803:Genes, Immunoglobulin; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D007136:Immunoglobulins / Q000276:immunology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D009113:Muramidase / Q000276:immunology; D010641:Phenotype; D013154:Spleen / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D000906:Antibodies; D000941:Antigens; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D008070:Lipopolysaccharides; C421864:hen egg lysozyme; D009113:Muramidase; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase