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.

中文翻译：

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通过免疫印迹可靠、灵敏地检测糖胺聚糖链

复杂聚糖在许多生物过程中发挥着至关重要的作用，从细胞内信号传导和器官发育到肿瘤生长。通常通过将聚糖特异性抗体应用于细胞和组织来评估聚糖表达。然而，聚糖特异性抗体经常在免疫印迹上显示大量条带，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策略来解决这个问题，该策略利用免疫反应增强技术来大大提高基于聚糖的免疫印迹的速度和质量。作为代表性案例研究，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，并使用单克隆抗体 CS-56 作为探针。我们发现，抗体与其抗原性 CS-GAG 链预孵育可区分真阳性信号和假阳性信号。我们成功地将这一策略应用于 10E4，一种单克隆抗硫酸乙酰肝素 GAGs (HS-GAGs) 抗体，其中真阳性信号通过膜上的化学 HS-GAG 解聚得到证实。这一证据表明，聚糖特异性抗体可以在免疫印迹上生成清晰、令人信服的数据，并具有高度可复制的结果，这为糖生物学生命科学研究的许多方面开辟了新的机会。