Cell-microarray analysis of antigen-specific B-cells: Single cell analysis of antigen receptor expression and specificity
Cell-microarray analysis of antigen-specific B-cells: Single cell analysis of antigen receptor expression and specificity
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DOI：
10.1002/cyto.a.20471
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发表时间：
2007-11-01
期刊：
影响因子：
3.7
通讯作者：
Muraguchi, Atsushi
中科院分区：
文献类型：
--
作者：
Tajiri, Kazuto;Kishi, Hiroyuki;Muraguchi, Atsushi
作者之前开发了一种细胞微阵列系统，通过监测单细胞水平的细胞内 Ca2+ 来有效检测抗原特异性 B 细胞。在这里，他们提出了一种使用细胞微阵列系统检测抗原特异性 B 细胞的新方法。为了检测抗原特异性 B 细胞，他们将活淋巴细胞排列在芯片上，用荧光标记的非特异性蛋白质对细胞进行染色，并用荧光扫描仪对其进行分析，以检测与 B 细胞结合的非特异性蛋白质。然后他们用荧光标记的抗原对细胞进行染色，并用扫描仪对其进行分析。用特异性抗原染色但未用非特异性蛋白质染色的细胞被确定为抗原特异性 B 细胞并收获。通过单细胞 RT-PCR 从回收的 B 细胞中扩增抗体 cDNA，插入表达载体中，并通过 ELISA 检查其特异性。他们可以使用在 B 细胞表面表达鸡蛋溶菌酶抗体的转基因小鼠，在模型系统中以 0.01% 的频率检测抗原特异性 B 细胞。他们应用该系统直接检测接种HBs-Ag疫苗的志愿者外周血中的乙型肝炎病毒表面抗原（HBs-Ag）特异性B细胞，并成功生产了HBs-Ag特异性单克隆抗体。这个新颖的系统使我们能够识别频率非常低的人类抗原特异性 B 细胞，并且是探索候选抗体疗法的强大工具。 (c) 2007 年国际分析细胞学学会。
The authors previously developed a cell-microarray system that effectively detects antigen-specific B-cells by monitoring intracellular Ca2+ at single cell levels. Here they present a novel method to detect antigen-specific B-cells using cell-microarray system. To detect antigen-specific B-cells, they arrayed live lymphocytes on a chip, stained cells with fluorescence-labeled nonspecific proteins, and analyzed them with a fluorescence scanner to detect nonspecific protein binding to B-cells. They then stained cells with fluorescence-labeled antigen and analyzed them with the scanner. Cells stained with specific antigen, but not with nonspecific proteins, were determined as antigen-specific B-cells and harvested. Antibody cDNA was amplified from retrieved B-cells by single-cell RT-PCR, inserted into expression vectors, and was examined for its specificity by ELISA. They could detect antigen-specific B-cells at a frequency of 0.01% in a model system using transgenic mice that express antibody to hen-egg lysozyme on the surface of B-cells. They applied this system to directly detect hepatitis B virus surface-antigen (HBs-Ag)-specific B-cells from peripheral blood in HBs-Ag-vaccinated volunteers and succeeded in producing HBs-Ag-specific monoclonal antibody. This novel system allows us to identify human antigen-specific B-cells of very low frequency and is a powerful tool to explore the candidates of antibody therapeutics. (c) 2007 International Society for Analytical Cytology.