Biotechnology Research Institute for Drug Discovery (BRD), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba 305-8577, Japan; JST PRESTO, Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Biotechnology Research Institute for Drug Discovery (BRD), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
J Biosci Bioeng. 2020 Feb;129(2):215-222. doi: 10.1016/j.jbiosc.2019.08.003. Epub 2019 Aug 28.
Human pluripotent stem cells (hPSCs) are considered ideal cell sources for regenerative medicine, but their clinical and industrial applications are hindered by their tumorigenic potential. We previously identified an hPSC-specific lectin, rBC2LCN, that recognizes the podocalyxin glycoprotein secreted by undifferentiated hPSCs into the culture media. Using biotinylated rBC2LCN and a peroxidase-labeled R-10G antibody, we developed a sandwich assay for the detection of tumorigenic hPSCs. In this assay, the lectin is randomly immobilized on streptavidin-coated microplates to capture hPSC-derived podocalyxin. In the present study, rBC2LCN was genetically fused with polystyrene-binding peptides (PS-tags) for direct, site-specific, and oriented immobilization on polystyrene microplates. rBC2LCN lectins fused with PS-tags at the C-terminus were successfully overexpressed as a soluble form in Escherichia coli and then purified by affinity chromatography. We optimized the various parameters (protein and NaCl concentration, buffer pH, and blocking agents) of the sandwich assay by using PS-tagged rBC2LCN and the R-10G antibody. Finally, the lower limit of detection (LLOD) of the sandwich assay for hPSCs was examined. The LLOD was 2.2-fold lower than that achieved with the previous method. Considering that the developed method does not require the precoating of polystyrene microplates with streptavidin, it provides a cost-effective approach for the highly sensitive detection of hPSCs residing in hPSC-derived cell therapeutics.
人多能干细胞（hPSCs）被认为是再生医学的理想细胞来源，但它们的临床和工业应用受到其致瘤潜能的阻碍。我们之前发现了一种 hPSC 特异性凝集素 rBC2LCN，它可以识别未分化的 hPSCs 分泌到培养基中的足细胞蛋白聚糖糖蛋白。我们使用生物素化的 rBC2LCN 和过氧化物酶标记的 R-10G 抗体，开发了一种用于检测致瘤性 hPSC 的三明治检测法。在该检测法中，凝集素随机固定在链霉亲和素包被的微孔板上，以捕获 hPSC 来源的足细胞蛋白聚糖。在本研究中，rBC2LCN 与聚苯乙烯结合肽（PS-标签）进行基因融合，用于在聚苯乙烯微孔板上进行直接、特异性和定向固定。C 末端融合有 PS-标签的 rBC2LCN 凝集素在大肠杆菌中成功地以可溶性形式过表达，然后通过亲和层析进行纯化。我们通过使用 PS 标记的 rBC2LCN 和 R-10G 抗体优化了三明治检测法的各种参数（蛋白和 NaCl 浓度、缓冲液 pH 值和封闭剂）。最后，我们检查了三明治检测法检测 hPSC 的最低检测限（LLOQ）。该检测法的 LLOD 比以前的方法低 2.2 倍。考虑到该方法不需要在聚苯乙烯微孔板上预涂链霉亲和素，它为 hPSC 衍生细胞治疗中残留的 hPSC 的高灵敏度检测提供了一种具有成本效益的方法。
