Efficient production of human recombinant antibodies against hepatitis virus using a novel micro-well array system

使用新型微孔阵列系统高效生产抗肝炎病毒的人重组抗体

• 批准号: 15390312
• 负责人: MURAGUCHI Atsushi
• 金额: $ 9.28万
• 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-15390312/
• 关键词: micro-well-array system; hepatitis virus; recombinant antibody; antibody-medicine; neutralizing antibody; 中和能; 肝炎ウイルス; Bリンパ球

Although infectious diseases caused by pathogenic microbes seemed extraordinarily lessened after the development of various drug discovery, newly emerging infectious diseases, such as worldwide-spreading AIDS or SARS broken out in Asia, have been a threat to humans. Antibodies (Abs) have been long expected as a desirable therapeutic reagent against the pathogenic microbe, their use has been extremely limited because of the difficulties to efficiently generate humanized monoclonal Abs to various micobes. In the present project, we have tried to develop a novel micro-well-array system to efficiently find the antigen (Ag)-specific human B cells and applied this sytem to produce human monoclonal Abs against hepatitis B virus-surface antigen (HbsAg).We have developed a micro-well-array chip consists of highly integrated micro chambers (250,000 wells in half square centimeter) on a silicon chip, of which chambers were designed to a size of a single B cell. To detect Ag-specific B cells, intr … More acellular Ca2^+ indicator, Fluo-4, was introduced to the cells as a marker for B cell activation via B cell receptor (BCR), and the increases in (Ca2^+)i were detected by the customized DNA-array.We applied this system to produce human monoclonal Abs against HbsAg. B cells from healthy volunteers who had been immunized with HBsAg were stimulated with HBsAg on a chip and the responding cells were detected by the scanner and harvested by micromanipulation. The cDNA of variable regions for Ig Hand L chains were cloned from the harvested B cells and the recombinant Abs were generated in 293T cells. The ability to bind to HbsAg or to neutralize the HB virus activity was assessed by ELISA or in vivo mouse model, respectively.As a result, we produced several human recombinant Abs with high binding affinity to HbsAg, as well as high neutralizing activity. This micro-well array system should be a high throughput system for producing human monoclonal Abs against various infectious microbes and contribute to discover antibody-medicine against newly emerging infectious diseases. Less

尽管随着各种药物的开发，由病原微生物引起的传染病似乎已大大减少，但新出现的传染病，例如全球范围内传播的艾滋病或亚洲爆发的非典型肺炎，长期以来一直对人类构成威胁。作为一种理想的针对病原微生物的治疗试剂，它们的用途受到了极大的限制，因为很难有效地产生针对各种微生物的人源化单克隆抗体。新型微孔阵列系统能够有效地发现抗原（Ag）特异性的人B细胞，并应用该系统生产针对乙型肝炎病毒表面抗原（HbsAg）的人单克隆抗体。我们开发了微孔阵列芯片由硅芯片上高度集成的微室（半平方厘米内有 250,000 个孔）组成，其中的室被设计成单个 B 细胞的大小，以检测 Ag 特异性 B 细胞。 intr ... 更多非细胞 Ca2^+ 指示剂 Fluo-4 被引入细胞中，作为通过 B 细胞受体 (BCR) 激活 B 细胞的标记物，并通过定制的 DNA- 检测到 (Ca2^+)i 的增加我们应用该系统生产针对 HbsAg 的人类单克隆抗体，这些 B 细胞来自已接种 HBsAg 的健康志愿者，并用芯片上的 HBsAg 进行刺激，并通过扫描仪检测到有反应的细胞。从收获的 B 细胞中克隆 Ig Hand L 链可变区的 cDNA，并在 293T 细胞中产生重组抗体。结果，我们生产了几种对 HbsAg 具有高结合亲和力以及高中和活性的人重组抗体。成为生产针对各种传染性微生物的人单克隆抗体的高通量系统，并有助于发现针对新出现的传染病的抗体药物。

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.

Kondo S., et al.: "Possible involvement of glial cell line-derived neurotrophic factor (GDNF) and its recentor, GFR α1, 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) 及其近期因子 GFR α1 可能参与胸腺细胞的存活和成熟”Eur.J.Immunol.. 33. 2233-2240 (2003 ）

Kawakami T., et al.: "Proteomic approach to apoptotic thymus maturation"J.Chromatography B.. 787. 223-229 (2003)

Kawakami T.等人：“凋亡胸腺成熟的蛋白质组学方法”J.Chromatography B.. 787. 223-229 (2003)

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