| Project/Area Number |
18390288
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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 | University of Toyama |
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Principal Investigator |
MURAGUCHI Atsushi University of Toyama, Graduate School of Medicine and Pharmaceutical Science for Research, Professor (20174287)
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| Co-Investigator(Kenkyū-buntansha) |
KISHI Hiroyuki University of Toyama, Graduate School of Medicine and Pharmaceutical Science for Research, Associate Professor (60186210)
KITAJIMA Isao University of Toyama, Graduate School of Medicine and Pharmaceutical Science for Research, Professor (50214797)
OZAWA Tatsuhiko University of Toyama, Graduate School of Medicine and Pharmaceutical Science for Research, Associate Professor (10432105)
本多 立 富山大学, 大学院医学薬学研究部, 助手 (90377250)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥17,000,000 (Direct Cost: ¥15,500,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2007: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2006: ¥10,500,000 (Direct Cost: ¥10,500,000)
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| Keywords | infectious disease / antibody therapy / lymphocyte microarray chip / antibody gene / recombinant antibody |
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| Research Abstract |
We developed a single-cell microarray system to analyze cellular response of individual lymphocytes using microwell array chips, with over 230,000 microwells that can accommodate only single-lymphocytes. We also developed new methods for(1)detection of Ag-specific B cells with Ca^2+ response and Ag-binding, and (2) detection of Ab-producing B cells by Fluorescence-linked immuno-spot (FLTSPOT) assay. Since the address of each lymphocyte on the chip is fixed, we could easily recover them using a micromanipulator. Antibody cDNA could be augmented from each single lymphocyte by RT-PCR and could be expressed in cells. By applying this system, we successfully obtained Abs for type B hepatitis virus surface antigen (HBs-Ag) as well as influenza virus from peripheral blood lymphocytes of HBs-Ag-and influenza-HA immunized volunteers. The new methods for detection of Ag-specific B cells is following; (1) Simultanous Ca^2+ response and Ag-binding assay in which B-cells were applied on the chip an
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d stained with non-specific protein-Cy5, followed by Antigen-Cy5, and fluorescence of cells was monitored with the Cell Scanner. This method enabled us to efficiently detect Ag-specific B cells with low frequency of false-positive Ag-specific B-cells.(B) FLTSPOT Assay in which anti-Ig-Ab was coated on the surface of microwell array chip and cells containing Ab-secreting cells were added to the chip, and Ag-specific Ab-secreting cells were detected using fluorescence-labeled Ag. Then Ag-specific Ab-secreting cells were retrieved from the chip, Ab cDNA was amplified with RT-PCR and recombinant Abs were produced. In the case of HBs antigen/Influenza-HA, enriched CD138^+ human lymphocytes were cultured shortly on chips and Ab-secreting cells were detected using biotinylated-antigens, followed by streptavidine-Cy3. V_H and V-L cDNAs cloned from single antibody-secreting cells were transfected into 293T cells and recombinant Abs were generated. Ag-specificity of antibodies were examined with ELISA in the presence of soluble antigens. Less
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