title: Antigen-based immunofluorescence analysis of B-cell targeting: advanced technology for the generation of novel monoclonal antibodies with high efficiency and selectivity.
abstract: On the basis of immunofluorescence analysis, an entire pathway of B-cell targeting was successfully identified, which can drive selective production of monoclonal antibodies with high efficiency and selectivity. The technique comprises three critical steps, antigen-based preselection of B lymphocytes, formation of antigenselected B lymphocyte and myeloma cell complexes, and selective fusion of B-cell-myeloma cell complexes with electrical pulses. Intriguingly, expression of surface immunoglobulin receptors on B lymphocytes was recognized even after immunization in vitro. The number of the antigen-selected B lymphocytes after in vitro immunization was in fact higher than that obtained after in vivo immunization, suggesting that such shortterm immunization is applicable for B-cell targeting. Immunofluorescence analysis revealed the targeting technique to demonstrate fivefold to tenfold higher efficiency for formation of hybridoma cells than a polyethylene glycol (PEG)-mediated method. This efficiency is in good agreement with production of hybridoma cells secreting desired monoclonal antibodies determined by an enzyme-linked immunosorbent assay (ELISA). The addition of a low concentration of PEG brought about enhanced fusion efficiency and reduced cell damage at even electric intensities as high as 4.0 kV/cm and 5.0 kV/cm. Here we demonstrate that immunofluorescence analysis can successfully clarify an entire pathway of B-cell targeting and provide advantages over the PEG-mediated method. This advanced technology may be applicable for rapid production of monoclonal antibodies based on in vitro immunization.
authors.affiliation: Division of Chemistry for Materials, Graduate School of Engineering, Mie University, Mie, Japan. tomita@chem.mie-u.ac.jp
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tomita
authors.forename: Takatomi
authors.initials: T
authors.lastname: Fukuda
authors.forename: Akira
authors.initials: A
authors.lastname: Ozu
authors.forename: Kei-ichi
authors.initials: K
authors.lastname: Kimura
authors.forename: Tian Yow
authors.initials: TY
authors.lastname: Tsong
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Yoshimura
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000276:immunology*; D002459:Cell Fusion / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D004526:Efficiency; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006825:Hybridomas / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D011092:Polyethylene Glycols / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D011092:Polyethylene Glycols