Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 486, Issue 4, Pages 1077-1082Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2017.03.165
Keywords
FLAG peptide; Monoclonal antibody; Chimeric antibody; Flowcytometry; Immunohistochemistry
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Funding
- MEXT/JSPS KAKENHI Grant [22116001, 22116002, 15H05901, 15H05904, 15H04708, 15K08316, 15K19032, 22860223, 24590386, 2546037]
- Naito Foundation
- Ono Medical Research Foundation
- Uehara Memorial Foundation
- Mitsubishi Foundation
- Nakatomi Foundation
- Talceda Science Foundation
- Foundation of Strategic Research Projects in Private Universities from the MEXT [S1311011]
- Institute for Environmental and Gender-Specific Medicine
- Grants-in-Aid for Scientific Research [15K08316, 15H05904, 15KK0320, 15K19032, 24590386, 16K08596] Funding Source: KAKEN
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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 fiowcytometry 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 fiowcytometry 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. (C) 2017 Elsevier Inc. All rights reserved.
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