1996 Fiscal Year Final Research Report Summary
Human monoclonal antibodies to Entamoeba histolytica prepared by phage display
| Project/Area Number |
07670292
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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 |
寄生虫学(含医用動物学)
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| Research Institution | Tokai University |
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Principal Investigator |
TACHIBANA Hiroshi Tokai University School of Medicine, Department of Infectious Diseases, Associate Professor, 医学部, 助教授 (10147168)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Entamoeba histolytica / monoclonal antibodies / Fab / phage display / expression vector |
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| Research Abstract |
Human monoclonal antibodies (Feb) to Entamoeba histolytica were prepared by a phage display system. In addition, for evaluating the expression system, a mouse Fab was also prepared from the RNA of hydridoma cells.
Mouse RNA was isolated from hybridoma cells secreting monoclonal antibody EH3015 to E histolytica, and the genes coding for the light chain and the Fd region of the heavy chain of the IgG molecule were amplified by RT-PCR.The PCR products were ligated with an expression vector pRPLS/Fab-I.Since this vector was originally prepared for a phage display system, gene III was removed for the production of soluble Fab in Escherichia coli. After gene expression, the E.coli were sonicated and the supernatant analyzed. The presence of Fab against E.histolytica was confirmed by an indirect immunofluorescence antibody test, ELISA,and Western blot analysis.
The RNA of lymphocytes isolated from the peripheral blood of E.histolytica-infected individuals was purified. By RT-PCR,genes coding for the light chain and the Fd region of the heavy chain of the human IgG molecule were amplified, and the PCR products ligated with the expression vector. The selection of phage clones expressing Fab was by panning in ELISA wells treated with crude antigens of E.histolytica. After removal of gene III from the vector, soluble Fabs were produced by Escherichia coli. When the supernatant of sonicated E.coli was analyzed by ELISA,42% of the clones examined showed production of human Fab to E.histolytica.
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Research Products
(8 results)
