Epitope Mapping of Anti-Diacylglycerol Kinase Zeta Monoclonal Antibody DzMab-1 for Immunohistochemical Analyses

Abstract

The diacylglycerol kinases (DGKs) catalyze the phosphorylation of the cell membrane lipid diacylglycerol (DG), which is important in lipid biochemistry and signal transduction into phosphatidic acid. DG-mediated signal transduction downstream of the T cell receptor has been reported to be terminated by DGKζ, 1 of 10 DGK isoforms in most cases. We previously established an anti-DGKζ monoclonal antibody (mAb) DzMab-1 (rat IgG₁, kappa), which reacts with both mouse DGKζ and human DGKζ (hDGKζ). In this study, we characterized the binding epitope of DzMab-1 using Western blotting, and found that Met1 and Pro3 residues of hDGKζ are important for facilitating DzMab-1 binding to hDGKζ. Furthermore, DzMab-1 was shown to be useful for immunohistochemical analyses for formalin-fixed paraffin-embedded HeLa cells. These findings could be applied for the production of more functional anti-hDGKζ mAbs.