Epitope mapping of an anti-diacylglycerol kinase delta monoclonal antibody DdMab-1

Abstract

Diacylglycerol kinase δ (DGKδ) is a type II DGK, which catalyzes diacylglycerol phosphorylation to produce phosphatidic acid. DGKδ is expressed in several types of tissues and organs including the stomach, testis, bone marrow, and lymph node. Here, we established an anti-human DGKδ (hDGKδ) mAb, DdMab-1 (mouse IgG 2a , kappa), which is useful for Western blot analysis. We also introduced deletion or point mutations to hDGKδ, and performed western blotting to determine the binding epitope of DdMab-1. DdMab-1 reacted with the dN670 mutant, but not with the dN680 mutant, indicating that the N-terminus of the DdMab-1 epitope is mainly located between amino acids 670 and 680 of the protein. Further analysis using point mutants demonstrated that R675A, R678A, K679A, and K682A mutants were not detected, and V680A was only weakly detected by DdMab-1, indicating that Arg675, Arg678, Lys679, Val680 and Lys682 are important for binding of DdMab-1 to hDGKδ.