| Project/Area Number |
11670820
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Dermatology
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| Research Institution | Toyama Medical and Pharmaceutical University |
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Principal Investigator |
MATSUI Chihiro TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY, hospital Assist Professor, 附属病院, 講師 (10181679)
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| Co-Investigator(Kenkyū-buntansha) |
MOROHASHI Masaaki TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY Faculty of Medicine Professor, 医学部, 教授 (50018719)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Laminin / Cicatricial pemphigoid / 瘢痕性類天庖瘡 / 組み換え蛋白 |
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| Research Abstract |
Cicatricial pemphigoid is autoimmune bullous disease that primarily affects mucosal tissues. Recent studies have identified that CP patients have IgG autoantibodies recognize several basement membrane proteins including laminin5 (Lain5). To further understand the pathophysiology of blister formation in these patient, we have sought to identify the specific epitope(s) targeted by their autoantibodies. Since bacterial fusion proteins that contain LAM α3 amino acid sequence do not react with patient sera in immunoblotting study, we used recombinant peptides made by in vitro transcription/ translation reaction for immunoprecipitation. The peptides that contain II/ I domain of LAM α3 were precipitated by patient sera and conformational epitope seems to be necessary for Antigen-Antibody bindings. Though IV domain of LAM α3 has be reported crucial for keratinocyte attachment, II/ I domain also might be imperative.
Since radioisotope immunoprecipitation (RI-IP) has high sensitivity to detect anti Lam5 autoantibody, we have examined CP patient sera with this assay. However autoradiography is time-consuming and the use of radioisotpe is restricted. Therefore we tried to develop the assay in which radioisotope are not used to detect autoantibodies. Sera from CP patients that were diagnosed by RI-IP were incubated with solubilized SCC25 cell fraction. The precipitated proteins were subjected to immunobloting. The blots were incubated with anti β3 subunit of Lam5 rabbit serum as first antibody and developed by ECL kit. Anti β3 antibody could detect precipitated subunit of Lam5 by patients' sera on nitrocellulose membrane. The sensitivity of non radioisotope immunoprecipitation assay is lower than RI-IP and treatment to enhance sensitivity (such as biotylation of cellular protein) is now in progress.
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