Analysis of Anti-mitochondrial Antibody Producing B Cell Repertoire in Primary Biliary Cirrhosis
| Project/Area Number | 03670326 |
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
内科学一般
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
ISHIBASHI Hiromi KYUSHU UNIVERSITY MEDICINE Lecturer, 医学部, 講師 (80127969)
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| Co-Investigator(Kenkyū-buntansha) |
KUDO Jiro KYUSHU UNIVERSITY MEDICINE Assist. Prof., 医学部, 助手 (90148940)
HIRATA Yasuhiko KYUSHU UNIVERSITY MEDICINE Assist. Prof., 医学部, 助手 (10218787)
NAKAMURA Minoru KYUSHU UNIVERSITY MEDICINE Assist. Prof., 医学部, 助手 (40217906)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed(Fiscal Year 1992)
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| Budget Amount *help |
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1992 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1991 : ¥1,000,000 (Direct Cost : ¥1,000,000)
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| Keywords | primary biliary cirrhosis, PBC / anti-mitochondrial antibody, AMA / B lymphocyte / antibody production / autoimmune disease / autoantibody / pyruvate dehydrogenase complex, PDC / 2-oxoglutarate dehydrogenase complex, OGDC / 2ーオキソグルタル酸脱水素酵素複合体 |
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| Research Abstract |
We performed an analysis of B cell repertoire in peripheral blood of patients with primary biliary cirrhosis (PBC) and cloning of B lymphocytes producing human monoclonal antibodies to pyruvate dehydrogenase complex (PDC) and anti-oxoglutarate dehydrogenase complex (OGDC) using highly purified PDC and OGDC. As a result we obtained the following findings;
1. Enzyme linked immunosorbent assay (ELISA) was established to assay anti-PDC and OGDC antibodies in sera of patients with PBC.
2. Analysis of positivity of anti-PDC and OGDC antibodies in patients with PBC and other hepatic, non-hepatic diseases including collagen diseases revealed that anti-PDC and OGDC antibodies were specific for PBC.
3. Frequency of B lymphocytes producing anti-PDC was calculated to be 0-2% of the total IgG producing B lymphocytes in peripheral blood of patients with PBC.
4. By comparing the frequency of B lymphocytes producing anti-PDC and anti-OGDC antibodies with titers of these antibodies and histological finding
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s, it was revealed that the frequency of B lymphocytes producing anti-PDC and anti-OGDC antibodies paralleled with the degree of infiltrating lymphocytes and the disease activity.
5. Three B lymphocyte clones producing human monoclonal antibodies (mAb) were established from peripheral B lymphocytes of a patient with PBC. Peripheral B lymphocytes from a patient with primary biliary cirrhosis were infected with Epstein-Barr virus(EBV), and EBV-transformed B lymphocytes producing large amounts of IgG antibodies to pyruvate dehydrogenase complex (PDC) were selected, expanded and fused with human mouse heteromyeloma cell line - F3B6. The resulting EBV-transformed B cell hybrids were repeatedly cloned by limiting dilution and three stable hybridoma clones producing human mAb to PDC were generated. These mAb, designated M18GP8, M37GP11 and M82GP8, specifically bound to PDC with high affinity. These three mAb stained the mouse stomach/kidney cryostat sections in a typical immunofluorescence pattern of anti-mitochondrial antibody. Furthermore, the enzymatic activity of PDC was almost completely inhibited by the three mAb. Western blotting analysis revealed that M18GP8 and M82GP8 reacted with only PDC-E2 in contrast to M37GP11 which reacted with both PDC-E2 and protein X.The binding of monoclonal antibody M37GP11 to solid phase PDC was partially inhibited by two different synthetic peptides corresponding to both the inner and outer lipoyl binding domains of PDC-E2. These mAb to PDC generated from a patient with PBC, would be a valuable tool to study the B cell autoepitopes in PDC and the mechanism of autoantibody production in PBC.
6. One B lymphocyte clone producing human mAb to OGDC was also established and the mAb was characterized for its H chain subclass, L chain, specificity and affinity to OGDC, inhibition of enzyme activity and immunoglobulin gene. Less
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Report
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Research Products
(6results)
