O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3′,5,5′-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.

O-GlcNAcylation是一种动态的，可逆的，翻译后修饰，可调节许多细胞过程。O-GlcNAc转移酶（OGT）是转移N的唯一酶-乙酰氨基葡萄糖从尿苷二磷酸（UDP）-GlcNAc到胞质和细胞核蛋白的选定丝氨酸/苏氨酸残基。OGT活性异常与多种疾病有关，提示OGT是一种新型治疗靶标。在这项研究中，我们创建了一种新的基于酶联免疫吸附测定（ELISA）的方法来检测OGT活性。首先，将酪蛋白激酶II（CKII）（一种众所周知的OGT底物）包被到ELISA板上。其次，使用针对O-GlcNAc的抗体，然后使用辣根过氧化物酶（HRP）标记的二抗，检测由OGT从UDP-GlcNAc转移至CKII的GlcNAc。最后，用3,3'，5,5'-四甲基联苯胺（TMB）作为HRP的底物，检测了CKII的O-GlcNAcylation水平，该水平反映了OGT的活性。根据一系列优化实验，选择RL2抗体进行O-GlcNAc检测，并确定CKII，OGT和UDP-GlcNAc的浓度。使用商用OGT抑制剂ST045849来验证系统的功能。总之，这项研究显示了一种可用于检测OGT活性和筛选OGT抑制剂的方法。