Human natural killer-1 (HNK-1) epitope, a highly-expressed glycan in the nervous system, is critical for normal synaptic plasticity and spatial learning. HNK-1 epitope modifies N-glycans on several neural glycoproteins, and also modifies O-mannosyl glycans. A branching enzyme for O-mannosyl glycans (GnT-IX, Core M2 synthase) exhibits brain-specific expression, and the product core M2 glycans are also limited to the brain. In a previous study, we showed that cuprizone-induced demyelination increased HNK-1-capped core M2 glycan expression, while GnT-IX deficiency ameliorated demyelination, suggesting that these glycans could be useful diagnostic markers for demyelination status and act as therapeutic targets. Nevertheless, a lack of appropriate detection tools hampered further analysis of HNK-1-capped O-mannosyl glycans. In the present study, we chemoenzymatically synthesized HNK-1-capped core M2 glycans for antibody production, and confirmed that the resulting immune sera reacted with HNK-1-capped core M2 glycans. We then examined several HNK-1-related antibodies, including the Cat-315 antibody, for reactions with HNK-1-capped core M2 glycans. Finally, we confirmed the increased HNK-1 epitope expression in demyelinated brains of cuprizone-fed mice.