Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide

• Published in: Biochemical and biophysical research communications Vol. 486; no. 4; pp. 1077 - 1082
• Main Authors: Ikeda, Koki, Koga, Tomoaki, Sasaki, Fumiyuki, Ueno, Ayumi, Saeki, Kazuko, Okuno, Toshiaki, Yokomizo, Takehiko
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.05.2017
• Subjects: Animals; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Chimeric antibody; FLAG peptide; Flowcytometry; Humans; Immunohistochemistry; Immunoprecipitation - methods; Mice; Monoclonal antibody; Oligopeptides - immunology; Protein Engineering - methods; Flowcytometry; Immunohistochemistry; Ab; Tg; GPCR; Monoclonal antibody; 5′ RACE; FLAG peptide; Chimeric antibody
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2017.03.165

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. •The FLAG peptide is used widely to investigate protein function and localization.•2H8 mouse Ab is sensitive for FLAG but yields nonspecific signals in mouse tissue.•The Fab region of mouse 2H8 Ab was fused with the Fc region of human IgG1.•Chimeric human 2H8 Ab detects FLAG without nonspecific signals in immunohistochemical analysis of mouse tissues.