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和抗原与受体的结合，启动淋巴细胞激活和分化。淋巴细胞激活通常通过96个井板中淋巴细胞的大量培养物的增殖或蛋白质分泌来监测。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做出反应。当可以在克隆水平上监测淋巴细胞激活时，将获得有关淋巴细胞激活的更精确的信息。为此，我们一直在开发用于检测克隆水平上抗原特异性淋巴细胞的系统。淋巴细胞装有钙指示剂，Fluo-4的荧光探针，并添加到硅芯片（2 cm x 2 cm）中，其中包含大约2 x 10^5微粒的阵列，其大小仅适合单个淋巴细胞。去除未进入微孔的残留细胞后，有70％至90％的井中含有单个淋巴细胞。激活前淋巴细胞的荧光由扫描仪监测。然后用抗原等兴奋剂激活淋巴细胞，并通过扫描仪监测淋巴细胞的荧光。通过比较激活之前和之后的荧光，使用微型手持剂从芯片中检测并从芯片中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。通过使用该系统，我们检测到抗原反应的人B淋巴细胞并获得了抗原特异性抗体。该系统对于开发抗体医学很简单且有用。它还将用于检测抗原特异性T淋巴细胞，可用于研究基础研究中的淋巴细胞激活以及临床应用

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

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.

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。

Activation of mouse RAG-2 promoter by Myc-associated zinc finger protein.

• DOI: 10.1016/j.bbrc.2004.03.159
• 发表时间: 2004-05
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Chun-xiao Wu;Wen-Pu Zhao;H. Kishi;Junichi Dokan;Zhe-xiong Jin;Xing-Cheng Wei;K. Yokoyama;A. Muraguchi

Kondo S., et al.: "Possible involvement of glial cell line-derived neurotrophic factor (GDNF) and its receptor, GFR α 1, in survival and maturation of thymocytes"Eur.J.Immunol.. 33. 2233-2240 (2003)

Kondo S. 等人：“神经胶质细胞系衍生的神经营养因子 (GDNF) 及其受体 GFR α 1 可能参与胸腺细胞的存活和成熟”Eur.J.Immunol.. 33. 2233-2240（ 2003）