Generation and application of new rat monoclonal antibodies against synthetic FLAG and OLLAS tags for improved immunodetection

Abstract

Previously, we prepared monoclonal antibodies (mAbs) by immunizing rats with the recombinant fusion proteins of mouse Langerin/CD207, which contained a flexible linker sequence from E. coli OmpF and a FLAG epitope. We found many of new rat mAbs were not reactive to mouse Langerin, and here we identify the epitopes of two of these IgG mAbs, L2 and L5, and assess their efficacy in various immunodetection methods. MAb L5 is a rat IgG mAb against the FLAG epitope, which detected both N-terminal and C-terminal FLAG tagged protein 2 to 8 times better than the conventional anti-FLAG mAb M2 by Western blot. For mAb L2, we found its epitope to be a 14 amino acid sequence SGFANELGPRLMGK which consisted of both sequences from the OmpF derived linker and mouse Langerin. This epitope sequence was named OLLAS (E. coli OmpF Linker and mouse Langerin fusion Sequence), and mAb L2 as mAb OLLA-2. When the OLLAS sequence was inserted into recombinant proteins at N-terminal, C-terminal, or internal sites, the OLLAS tag was detected by mAb OLLA-2 with very high sensitivity compared to other conventional epitope tags and anti-tag mAbs. MAb OLLA-2 recognized OLLAS tagged proteins with at least 100-fold more sensitivity than anti-FLAG M2 and anti-V5 mAbs in Western blot analyses. We also find the OLLAS epitope to be superior in immunoprecipitation and other immunodetection methods, such as fluorescent immunohistochemistry and flow cytometry. In the process, we successfully utilized the OLLAS epitope sequence as an internal linker for fusion between the engineered mAb and the antigen, and thus achieved improved immunodetection.