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

尽管在各种药物发现发生后，由致病性微生物引起的感染性疾病似乎却大大减少，但在亚洲爆发的新出现的感染（例如全球范围内的艾滋病或SARS）对人类构成威胁。长期以来，人们一直认为抗体（ABS）是针对致病性微生物的理想治疗试剂，由于难以有效地对各种微生物产生人源化的单克隆ABS，它们的使用非常有限。在本项目中，我们试图开发一种新型的微孔阵列系统，以有效地找到抗原（AG）特定的人类B细胞，并应用该系统对针对乙型肝炎病毒表面抗原（HBSAG）（HBSAG）产生人类单克隆ABS（HBSAG）。我们已经开发了由高度的Micro-Well-array Chip Chip Chors组成的一架平方（250 squars）（250 square）（250 square）（250 square）（250 square）（2500,000）将腔室设计为单个B单元的大小。为了检测Ag特异性B细胞，将其Inthm Intharm Ca2^+指示剂Fluo-4引入到细胞中，作为通过B细胞受体（BCR）激活B细胞激活的标记，并且（Ca2^+）I的增加（CA2^+）I被定制的DNA-Array.我们应用了该系统以生成人类单核腹腔抗HBSAG。用HBSAG免疫的健康志愿者的B细胞用芯片上的HBSAG刺激，并通过扫描仪检测到响应细胞，并通过微观渗透回收。从收获的B细胞中克隆了Ig手L链的可变区域的cDNA，并在293T细胞中产生重组ABS。通过ELISA或体内小鼠模型评估与HBSAG或中和HB病毒活性的能力。结果，我们产生了几种对HBSAG的高结合亲和力以及高中和活性的人类重组ABS。该微孔阵列系统应该是一种高吞吐量系统，用于针对各种感染性微生物产生人类单克隆ABS，并有助于发现针对新出现的感染的抗体中的抗体。较少的

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 ）

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

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)