Laboratory of Developmental Neurobiology, Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA.
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan.
Glycobiology. 2021 Feb 9;31(2):116-125. doi: 10.1093/glycob/cwaa060.
Complex glycans play vital roles in many biological processes, ranging from intracellular signaling and organ development to tumor growth. Glycan expression is routinely assessed by the application of glycan-specific antibodies to cells and tissues. However, glycan-specific antibodies quite often show a large number of bands on immunoblots and it is hard to interpret the data when reliable controls are lacking. This limits the scope of glycobiology studies and poses challenges for replication. We sought to resolve this issue by developing a novel strategy that utilizes an immunoreaction enhancing technology to vastly improve the speed and quality of glycan-based immunoblots. As a representative case study, we used chondroitin sulfate glycosaminoglycan (CS-GAG) chains as the carbohydrate target and a monoclonal antibody, CS-56, as the probe. We discovered that preincubation of the antibody with its antigenic CS-GAG chain distinguishes true-positive signals from false-positive ones. We successfully applied this strategy to 10E4, a monoclonal anti heparan sulfate GAGs (HS-GAGs) antibody, where true-positive signals were confirmed by chemical HS-GAG depolymerization on the membrane. This evidence that glycan-specific antibodies can generate clear and convincing data on immunoblot with highly replicable results opens new opportunities for many facets of life science research in glycobiology.
复杂糖链在许多生物过程中发挥着重要作用，从细胞内信号传递和器官发育到肿瘤生长。糖链的表达通常通过应用糖链特异性抗体来评估细胞和组织。然而，糖链特异性抗体在免疫印迹上经常显示出大量的条带，并且当缺乏可靠的对照时，很难解释数据。这限制了糖生物学研究的范围，并对复制提出了挑战。我们试图通过开发一种新的策略来解决这个问题，该策略利用免疫反应增强技术来极大地提高基于糖的免疫印迹的速度和质量。作为一个代表性的案例研究，我们使用硫酸软骨素糖胺聚糖 (CS-GAG) 链作为碳水化合物靶标和单克隆抗体 CS-56 作为探针。我们发现，抗体与抗原性 CS-GAG 链预先孵育可将真实阳性信号与假阳性信号区分开来。我们成功地将该策略应用于 10E4，一种单克隆抗肝素硫酸糖胺聚糖 (HS-GAG) 的抗体，其中真正的阳性信号通过膜上的 HS-GAG 解聚化学得到证实。这一证据表明，糖链特异性抗体可以在免疫印迹上产生清晰、令人信服的数据，并且结果具有高度可重复性，为糖生物学生命科学研究的许多方面开辟了新的机会。
