| Project/Area Number |
15390312
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
膠原病・アレルギー・感染症内科学
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| Research Institution | Toyama Medical and Pharmaceutical University |
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Principal Investigator |
MURAGUCHI Atsushi Toyama Medical and Pharmaceutical University, Department of Medicine, Professor, 医学部, 教授 (20174287)
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| Co-Investigator(Kenkyū-buntansha) |
KITAJIMA Isao Toyama Medical and Pharmaceutical University, Department of Medicine, Professor, 医学部, 教授 (50214797)
KISHI Hiroyuki Toyama Medical and Pharmaceutical University, Department of Medicine, Associate Professor, 医学部, 助教授 (60186210)
SUZUKI Masayasu Toyama University, Department of Engineering, Professor, 工学部, 教授 (70226554)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥14,500,000 (Direct Cost: ¥14,500,000)
Fiscal Year 2004: ¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 2003: ¥8,500,000 (Direct Cost: ¥8,500,000)
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| Keywords | 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 |
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| Research Abstract |
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
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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
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