Development of the method for detection of antigen-specific lymphocytes and antigen receptors using cell-microarray

开发利用细胞微阵列检测抗原特异性淋巴细胞和抗原受体的方法

• 批准号: 15390176
• 负责人: KISHI Hiroyuki
• 金额: $ 7.94万
• 依托单位: Toyama Medical and Pharmaceutical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390176/
• 关键词: Cell microarray; Antigen-specific lymphocytes; Antigen receptor gene; Antibody Medicine; マイクロウェル; アレイ; チップ; Bリンパ球; 抗体; カルシウム

Lymphocytes express antigen-specific receptors and binding of antigen to the receptors initiates lymphocyte-activation as well as differentiation. Lymphocyte-activation is usually monitored by the proliferation or the protein-secretion of bulk culture of lymphocytes in 96 well plates. At the clonal level, each lymphocyte expresses antigen-receptors with distinct specificity and responds to distinct epitopes in an antigenic molecule. When lymphocyte-activation could be monitored at the clonal level, more precise information on lymphocyte-activation will be obtained. To that end, we have been developing the system for detecting antigen-specific lymphocytes at the clonal level. Lymphocytes are loaded with fluorescent probe of calcium indicator, fluo-4, and added to a silicon chip (2 cm x 2 cm) containing array of about 2 x 10^5 microwells of which size is just fit for a single lymphocyte. After removing the residual cells that do not enter microwells, 70 to 90% of wells contain single lymphocytes. Fluorescence of lymphocytes before activation is monitored by a scanner. Then lymphocytes are activated with stimulants such as antigens and fluorescence of lymphocytes is monitored by the scanner. By comparing the fluorescence before and after activation, activated lymphocytes are detected and recovered from the chip using micromanipulator, and cDNA of antigen-receptors is recovered from a single lymphocyte. By using this system, we detected antigen-responding human B-lymphocytes and obtained antigen-specific antibodies. This system is simple and useful to develop antibody medicine. It will be also used to detect antigen-specific T-lymphocytes and useful to study lymphocyte-activation in basic research as well as clinical application

淋巴细胞表达抗原特异性受体，抗原与受体的结合启动淋巴细胞活化以及分化。淋巴细胞活化通常通过96孔板中大量培养的淋巴细胞的增殖或蛋白分泌来监测。在克隆水平上，每个淋巴细胞表达具有不同特异性的抗原受体，并对抗原分子中的不同表位产生应答。当可以在克隆水平上监测淋巴细胞活化时，将获得关于淋巴细胞活化的更精确的信息。为此，我们一直在开发用于在克隆水平上检测抗原特异性淋巴细胞的系统。将淋巴细胞用钙指示剂fluo-4的荧光探针加载，并添加到含有约2 x 10^5个微孔阵列的硅芯片（2 cm x 2 cm）中，微孔的大小刚好适合单个淋巴细胞。除去未进入微孔威尔斯的残留细胞后，70 - 90%的威尔斯孔含有单个淋巴细胞。淋巴细胞活化前的荧光由扫描仪监测。然后用刺激剂如抗原激活淋巴细胞，并通过扫描仪监测淋巴细胞的荧光。通过比较活化前后的荧光，使用显微操作器从芯片上检测和回收活化的淋巴细胞，并从单个淋巴细胞中回收抗原受体的cDNA。利用该系统，我们检测了抗原反应的人B淋巴细胞，并获得了抗原特异性抗体。该系统操作简单，对抗体药物的开发具有一定的实用价值。它也将用于检测抗原特异性T淋巴细胞，并在基础研究和临床应用中研究淋巴细胞活化

项目成果

バイオチップの最新技術と応用

生物芯片最新技术及应用

• 发表时间: 2004
• 作者: 岸 裕幸;他
• 通讯作者: 他

Liu Q.-L., et al.: "Heat-shock protein 70 binds caspase-activated DNase and enhances its activity in TCR-stimulated T cells"Blood. 102. 1788-1796 (2003)

Liu Q.-L. 等人：“热休克蛋白 70 结合 caspase 激活的 DNase，并增强其在 TCR 刺激的 T 细胞中的活性”血液。

Involvement of NF-κB in TGF-β-mediated suppression of IL-4 signaling.

NF-κB 参与 TGF-β 介导的 IL-4 信号传导抑制。

• 发表时间: 2004
• 期刊: Biochem.Biophys.Res.Commun. 313
• 作者: Yamamoto T.;et al.
• 通讯作者: et al.

RGA protein associates with a TRPV ion channel during biosynthesis and trafficking

• DOI: 10.1002/jcb.10775
• 发表时间: 2004-03-01
• 期刊: JOURNAL OF CELLULAR BIOCHEMISTRY
• 影响因子: 4
• 作者: Barnhill, JC;Stokes, AJ;Turner, H
• 通讯作者: Turner, H

Characterization of the proximal enhancer element and transcriptional regulatory factors for murine recombination activating gene‐2

• DOI: 10.1002/eji.200425185
• 发表时间: 2005-02
• 期刊: European Journal of Immunology
• 影响因子: 5.4
• 作者: Xing-Cheng Wei;Jun-ichi Dohkan;H. Kishi;Chun-xiao Wu;Sachiko Kondo;A. Muraguchi