Generation of chimeric high‐affinity monoclonal antibody against DYKDDDDK peptide (FLAG)

Abstract

DYKDDDDK peptide (FLAG) is a useful tool to investigate the function and localization of proteins whose antibodies are not available. Although several monoclonal antibodies (mAbs) for FLAG are commercially available, they sometimes fails to detect specific signals, rather detects a plenty of nonspecific signals upon the assays. We recently established a hybridoma that produces a high‐affinity mAb for FLAG (clone 2H8, Sasaki F, Anal Biochem. 2012). 2H8 antibody is sensitive enough to detect FLAG‐fused proteins by flowcytometry and immunoprecipitation. However, we observed nonspecific signals with 2H8 mAb in immunohistochemistry, because 2H8 mAb is mouse origin. In the present study, we tried to generate chimeric 2H8 mAb with Fc fragments derived from human immunoglobulin in order to reduce the nonspecific signals. We first cloned 5′ terminus cDNA (Fab region) of heavy and light chains of 2H8 mAb by 5′‐Rapid Amplification of cDNA End (5′‐RACE), and fused them to 3′ terminus cDNA (Fc region) of heavy and light chains of human IgG1. We next transiently transfected both chimeric plasmids into human embryonic kidney HEK293 cells with polyethyleneimine (PEI), and then medium was collected and subjected to flowcytometry. Human chimeric 2H8 mAb detected FLAG‐tagged protein. Taken together, we succeeded to generate human chimeric high‐affinity mAb against FLAG. We will further determine whether chimeric 2H8 decreases background signals with mouse tissues in the future.Support or Funding InformationThis work was supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) Nos. 22116001, 22116002, 15H05901, 15H05904, and 15H04708.