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

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Highlights

  • 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.

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.

Introduction

Epitope tagging techniques enable the detection of recombinant proteins for which specific antibodies (Abs) are not available [1]. Tagged proteins can be detected by various biochemical and cell biological techniques including flowcytometry, western blotting and immunofluorescent staining with Abs that are specific for each tag. Tagging systems are also widely applied for affinity purification and immunoprecipitation. High-affinity Abs against representative epitope tags, including the DYKDDDDK FLAG tag, influenza virus hemagglutinin (YPYDVPDYA) and c-myc (EQKLISEEDL) peptides are commercially available [2], [3], [4], [5]. The FLAG sequence is a hydrophilic tag that can be separated from the target protein by enterokinase and this tag is frequently used in various in vitro and in vivo experiments [6], [7], [8], [9]. Four anti-FLAG monoclonal Abs (mAbs), namely, calcium-dependent M1 and calcium-independent M2, M5, and L5 are now commercially available [10], [11], [12], [13]. However, unfortunately they are not sensitive enough to detect FLAG-fused proteins expressed at low levels. We previously established a novel hybridoma cell line that produces a high-affinity Ab against FLAG, namely, 2H8, which is sensitive enough to detect amino-terminal FLAG-tagged G protein-coupled receptors (GPCRs) and soluble proteins in crude preparations [14]. In addition, 2H8 mAb is able to immunoprecipitate FLAG-tagged GPCRs from cell lysates [7], [14]. Although 2H8 mAb is highly sensitive and specific for the FLAG sequence, it also gives rise to some background staining when used to detect FLAG-tagged proteins in transgenic (Tg) mice in vivo. This might be due to the mouse origin of 2H8 mAb. In the present study, we generated human-mouse hybrid chimeric 2H8 Ab, which has the antigen-specific Fab region from intact 2H8 mAb and an antigen-unrelated Fc region from human IgG1, and evaluated its efficacy in various biochemical and cell biological experiments.

Section snippets

Cell culture

2H8-producing hybridoma cells were maintained in Hybridoma-SFM (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS), 5% BM condimed H1 hybridoma cloning supplement (Sigma-Aldrich, St. Louis, MO), 2 mM l-glutamine and penicillin/streptomycin (P/S). Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified essential medium (DMEM) (Wako, Osaka, Japan) supplemented with 10% FBS and P/S. Chinese hamster ovary (CHO) cells were maintained in Ham's

Establishment of human-mouse chimeric 2H8, h2H8

To decrease the nonspecific background signal of 2H8 mAb in immunofluorescence staining experiments on mouse tissues, we tried to generate h2H8 Ab. Total RNA from 2H8 hybridoma cells was subjected to 5′-RACE and we successfully cloned 5′ end sequences of both heavy and light chains of 2H8 mAb (Genbank accession nos. LC199873 and LC199874, respectively). We then subcloned and fused the cDNA fragments for putative variable regions of both heavy and light chains of 2H8 mAb with the cDNA of the

Acknowledgements

We thank Drs. Junken Aoki and Asuka Inoue from Tohoku University for the plasmids encoding heavy and light chains of human IgG1 and helpful advices. This work was supported by MEXT/JSPS KAKENHI Grant Numbers, 22116001, 22116002, 15H05901, 15H05904, 15H04708, 15K08316, 15K19032, 22860223, 24590386, and 2546037, and grants from the Naito Foundation, the Ono Medical Research Foundation, the Uehara Memorial Foundation, the Mitsubishi Foundation, the Nakatomi Foundation and the Takeda Science

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