title: Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.
abstract: DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues.
authors.affiliation: Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Ikeda
authors.affiliation: Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan; Priority Organization for Innovation and Excellence, Program for Leading Graduate Schools "HIGO (Health Life Science: Interdisciplinary and Glocal Oriented) Program", Kumamoto University, Kumamoto, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Koga
authors.affiliation: Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Fumiyuki
authors.initials: F
authors.lastname: Sasaki
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Ueno
authors.affiliation: Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Kazuko
authors.initials: K
authors.lastname: Saeki
authors.affiliation: Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Okuno
authors.affiliation: Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan. Electronic address: yokomizo-tky@umin.ac.jp.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Yokomizo
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D051379:Mice; D009842:Oligopeptides / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D009842:Oligopeptides; C085706:FLAG peptide
keywords: Chimeric antibody; FLAG peptide; Flowcytometry; Immunohistochemistry; Monoclonal antibody