| Project/Area Number |
10670240
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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) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Entamoeba histolytica / human monoclonal anntibodies / lectin |
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| Research Abstract |
Despite the medical importance of Entamoeba histolytica, an effective vaccine to prevent amebiasis has not yet been developed. However, it has been reported that passive immunization with human anti-amebic antibodies obtained from patients with amebic liver abscess exerted a protective effect against amebic liver abscess formation in a mouse model. Therefore, such antibodies may be useful in attempts to reduce mortality from amebiasis by passive immunization. We have prepared recombinant human monoclonal antibody Fab fragments in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A) RNA was isolated from the lymphocytes and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase-polymerase chain reaction. Amplified DNA fragments were ligated with a plasmid vector pFab1-His2 and introduced into Escherichia coli. Bacterial colonies were transferred to nitrocellulose membranes and then screened for the production of antibodies by incubation with E. histolytica antigens followed by horseradish peroxidase-conjugated antibodies of the patient. One positive clone was selected for further analysis. The monoclonal Fab fragment reacted with 10 reference strains of E. histolytica, as shown by an indirect fluorescence antibody test using fixed trophozoites. In contrast, the antibody failed to react with other enteric protozoan parasites. Incubation of intact trophozoites with the antibody fragment localized the epitope on the cell surface. Western immunoblot analysis showed that the molecular mass of the E. histolytica antigen recognized by the monoclonal antibody Fab fragment was 260-kDa under nonreducing conditions. The Fab fragment significantly inhibited the adherence of E. histolytica to human erythrocytes.
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