De Jong M O, Rozemuller H, Bauman J G, Visser J W
Department of Hematology, Erasmus University Rotterdam, Netherlands.
J Immunol Methods. 1995 Jul 17;184(1):101-12. doi: 10.1016/0022-1759(95)00080-t.
The main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that binding of the biotinylated ligand to the receptor does not inhibit the subsequent interaction of biotin with fluorescently tagged avidin or streptavidin. Using interleukin-2 (IL-2), we compared the usefulness of various biotinylation reagents, NHS-biotin, S-NHS-biotin, S-NHS-LC-biotin, DBB and photobiotin, and developed optimal biotinylation conditions for the preparation of biologically active biotin-labeled IL-2 and the detection of IL-2 receptor expressing cells by flow cytometry. As determined by spot blot analysis, biotinylation of IL-2 was most efficient at the highest biotin-to-protein (B:P) ratio used. At a B:P ratio of 100, most of the biological activity of IL-2 was retained when S-NHS-LC-biotin was used. In contrast, most of the biological activity of IL-2 samples that were labeled with NHS-biotin or photobiotin was lost under these conditions. Biotin-labeled IL-2 preparations were tested in order to detect IL-2 receptors on IL-2 dependent CTLL-2 cells by flow cytometry after sequential staining with the biotinylated IL-2 and fluorescence tagged streptavidin. A high B:P ratio generally resulted in a high specific fluorescence intensity of the cells, particularly when S-NHS-LC-biotin was used as the biotinylation reagent. Biotin-IL-2 could also be used to detect IL-2 receptors expressed by lymphocytes in peripheral blood and bone marrow. Comparison of staining of lymphocytes with biotinylated IL-2 and an antibody against the IL-2 receptor alpha chain demonstrated that only a subset of the cells that showed a strong fluorescence signal after staining with biotinylated IL-2 expressed high numbers of the IL-2 receptor alpha chain. This is in agreement with the expression of functional IL-2 receptors on resting T cells and NK cells which do not express the alpha chain. After stimulation with PHA, virtually all lymphocytes expressed the alpha chain, whereas only part of these cells showed a strong fluorescence signal after staining with biotin-IL-2, while the rest of the cells had very low numbers of IL-2 binding sites. Our results demonstrate that, in addition to staining individual receptor subunits with antibodies, staining with biotinylated IL-2 is a useful indicator of functional IL-2 receptor expression.
使用生物素化配体通过流式细胞术研究异质细胞群体（如外周血或骨髓）上生长因子受体表达的主要前提条件是，生物素化配体保留其结合能力，并且生物素化配体与受体的结合不会抑制生物素随后与荧光标记的抗生物素蛋白或链霉抗生物素蛋白的相互作用。我们使用白细胞介素-2（IL-2）比较了各种生物素化试剂（NHS-生物素、S-NHS-生物素、S-NHS-LC-生物素、DBB和光生物素）的效用，并为制备具有生物活性的生物素标记的IL-2以及通过流式细胞术检测表达IL-2受体的细胞制定了最佳生物素化条件。通过斑点印迹分析确定，在使用的最高生物素与蛋白质（B:P）比例下，IL-2的生物素化效率最高。在B:P比例为100时，使用S-NHS-LC-生物素时IL-2的大部分生物活性得以保留。相比之下，用NHS-生物素或光生物素标记的IL-2样品在这些条件下大部分生物活性丧失。对生物素标记的IL-2制剂进行测试，以便在用生物素化的IL-2和荧光标记的链霉抗生物素蛋白进行顺序染色后，通过流式细胞术检测IL-2依赖性CTLL-2细胞上的IL-2受体。高B:P比例通常导致细胞具有高特异性荧光强度，特别是当使用S-NHS-LC-生物素作为生物素化试剂时。生物素化IL-2也可用于检测外周血和骨髓中淋巴细胞表达的IL-2受体。用生物素化的IL-2和抗IL-2受体α链抗体对淋巴细胞染色的比较表明，在用生物素化的IL-2染色后显示强荧光信号的细胞中，只有一部分表达大量的IL-2受体α链。这与静息T细胞和NK细胞上功能性IL-2受体的表达一致，这些细胞不表达α链。用PHA刺激后，几乎所有淋巴细胞都表达α链，而在用生物素化IL-2染色后，只有部分细胞显示强荧光信号，其余细胞的IL-2结合位点数量非常少。我们的结果表明，除了用抗体对单个受体亚基进行染色外，用生物素化的IL-2染色是功能性IL-2受体表达的有用指标。
