Single epitope multiple staining to detect ultralow frequency B cells
Single epitope multiple staining to detect ultralow frequency B cells
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DOI：
10.1016/s0022-1759(00)00352-5
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发表时间：
2001-03-01
影响因子：
2.2
通讯作者：
Cornall, RJ
中科院分区：
文献类型：
--
作者：
Townsend, SE;Goodnow, CC;Cornall, RJ
在这里，我们描述了一种新的方法，即单表位多重染色(SEMS)，用于从无关细胞的高背景中检测超低频靶细胞。在小鼠脾细胞样本中接种少量转鸡蛋白溶菌酶(HEL)特异性免疫球蛋白(Ig)的小鼠脾细胞。用转基因B细胞表达的同一表位的两种试剂对这些样品进行染色，这些试剂与两种不同的检测标记(FITC和生物素)连接。由于试剂的非特异性结合和细胞的概率分布，单一表位的双重染色使我们能够减少背景。我们也能够基于雪花只有一件事来检测细胞，那就是它们的抗原特异性。SEMS方法使我们能够重复检测到每一百万个细胞中低于一个细胞的频率的转基因细胞。除了检测和分离抗原特异性淋巴细胞外，SEMS还可用于提高许多基于荧光的应用的灵敏度，包括检测和高度特异地分离转基因细胞、转化细胞、干细胞、胎儿细胞或感染性生物。(C)2001 Elsevier Science B.V.保留所有权利。
Here we describe a method for detecting ultralow frequency target cells from within a high background of irrelevant cells by a novel method, single epitope multiple staining (SEMS). Samples of murine splenocytes were seeded with a low number of splenocytes from mice transgenic for a hen eggwhite lysozyme (HEL)-specific immunoglobulin (Ig). These samples were stained with two reagents specific for the same epitope expressed by the transgenic B cells, which had been conjugated to two different detectable labels (FITC and biotin). This dual staining of a single epitope allowed us to reduce the background due both to non-specific binding of reagents and to probabilistic distribution of the cells. We also were able to detect the cells based on snowing only one thing about them, namely, their antigen specificity. The SEMS method allowed us to reproducibly detect transgenic cells at frequencies below one cell in one million cells. SEMS could be used to increase the sensitivity of numerous fluorescence-based applications in addition to the detection and isolation of antigen-specific lymphocytes, including the detection and highly specific isolation of genetically modified cells, transformed cells, stem cells, fetal cells, or infectious organisms. (C) 2001 Elsevier Science B.V. All rights reserved.