| Project/Area Number |
10557051
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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 | 展開研究 |
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| Research Field |
内科学一般
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| Research Institution | Osaka University |
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Principal Investigator |
IWATANI Yoshinori Osaka Univ.Med.Scho, Professor, 医学部, 教授 (60168581)
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| Co-Investigator(Kenkyū-buntansha) |
小杉 厚 大阪大学, 医学部, 助教授 (90186685)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | immune function test / antibody-producing ell / membrane proteins cells producing soluble / cytokine-producing cell / image analysis / enzyme immunoassay / 細胞検出法 / 酸素抗体法 / 免疫機能検査 |
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| Research Abstract |
A direct counting method of single cells producing soluble factors, such as an ELISPOT assay, is frequently used in research of various fields. However, it takes a lot of time and effort to count spots formed in the assay, and furthermore subjective. Therefore, we tried to develop an automatic counter of single cells producing soluble factors by using an image analyzer. First, we set up an ELISPOT assay for lymphocytes producing anti-thyroid peroxidase autoantibodies, and make the formed spots clearer by data treatment of the image analyzer. Furthermore, we decreased the background staining of nitrocellulose membrane in the well of microplates by subtract the non-specific staining from the staining of the ELISPOT assay. Unfortunately, the trial to obtain the sum of spots with various sizes and density were failed, but the number of spots formed in the ELISPOT assay was correlated with the concentrations of anti-thyroid peroxidase autoantibodies in the supernatants of the wells in the assays. We could not set up the fluorescence SPOT assay for lymphocytes producing cytokines, because of the weak fluorescence intensity of spots. We set up an ELISPOT assay for lymphocytes producing soluble membrane proteins (sCD4 or sCD8) spontaneously. The number of sCD8-producing cells decreased during pregnancy, but that of sCD4-producing cells did not. In this study, we opened a way of developing an automatic analyzer for single immune cells producing soluble factors.
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