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Protection against invasive amebiasis by a single monoclonal antibody directed against a lipophosphoglycan antigen localized on the surface of Entamoeba histolytica.

Marinets A, Zhang T, Guillén N, Gounon P, Bohle B, Vollmann U, Scheiner O, Wiedermann G, Stanley SL, Duchêne M - J. Exp. Med. (1997)

Bottom Line: Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A.Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess.Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

View Article: PubMed Central - PubMed

Affiliation: Institute for Specific Prophylaxis and Tropical Medicine, A-1095 Vienna, Austria.

ABSTRACT
A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

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Immunoblot: membrane preparations from axenically cultured E. histolytica HM-1:IMSS (1), 200:NIH (2), HK-9  (3), and SFL-3 (4), xenically  grown E. histolytica SFL-3 (5),  axenically grown E. dispar  SAW760 (6), xenically grown E.  dispar SAW142 (7), and axenically grown T. vaginalis (8) were  separated by SDS-PAGE, blotted  onto nitrocellulose, and probed  with a 1:100 dilution of hybridoma supernatant from antibody  EH5 (A lanes) or buffer only (B  lanes). Bound antibodies were  detected with 125I-labeled sheep  anti–mouse antibodies.
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Figure 1: Immunoblot: membrane preparations from axenically cultured E. histolytica HM-1:IMSS (1), 200:NIH (2), HK-9 (3), and SFL-3 (4), xenically grown E. histolytica SFL-3 (5), axenically grown E. dispar SAW760 (6), xenically grown E. dispar SAW142 (7), and axenically grown T. vaginalis (8) were separated by SDS-PAGE, blotted onto nitrocellulose, and probed with a 1:100 dilution of hybridoma supernatant from antibody EH5 (A lanes) or buffer only (B lanes). Bound antibodies were detected with 125I-labeled sheep anti–mouse antibodies.

Mentions: Preliminary ELISA tests had shown that antibody EH5 was able to discriminate between E. histolytica and E. dispar (data not shown). Fig. 1 shows the immunoblot results obtained with membrane preparations from different xenically and axenically grown E. histolytica and E. dispar strains using antibody EH5. In addition, a membrane preparation from Trichomonas vaginalis, another protist parasite which is only distantly related to E. histolytica and E. dispar, was prepared and included. The immunoblots show strong reactivity for all E. histolytica strains of antigens migrating as a polydisperse band, whereas E. dispar bound to antibody EH5 weakly, and T. vaginalis hardly at all. In addition, the signals were stronger when the amebas had been grown axenically.


Protection against invasive amebiasis by a single monoclonal antibody directed against a lipophosphoglycan antigen localized on the surface of Entamoeba histolytica.

Marinets A, Zhang T, Guillén N, Gounon P, Bohle B, Vollmann U, Scheiner O, Wiedermann G, Stanley SL, Duchêne M - J. Exp. Med. (1997)

Immunoblot: membrane preparations from axenically cultured E. histolytica HM-1:IMSS (1), 200:NIH (2), HK-9  (3), and SFL-3 (4), xenically  grown E. histolytica SFL-3 (5),  axenically grown E. dispar  SAW760 (6), xenically grown E.  dispar SAW142 (7), and axenically grown T. vaginalis (8) were  separated by SDS-PAGE, blotted  onto nitrocellulose, and probed  with a 1:100 dilution of hybridoma supernatant from antibody  EH5 (A lanes) or buffer only (B  lanes). Bound antibodies were  detected with 125I-labeled sheep  anti–mouse antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199119&req=5

Figure 1: Immunoblot: membrane preparations from axenically cultured E. histolytica HM-1:IMSS (1), 200:NIH (2), HK-9 (3), and SFL-3 (4), xenically grown E. histolytica SFL-3 (5), axenically grown E. dispar SAW760 (6), xenically grown E. dispar SAW142 (7), and axenically grown T. vaginalis (8) were separated by SDS-PAGE, blotted onto nitrocellulose, and probed with a 1:100 dilution of hybridoma supernatant from antibody EH5 (A lanes) or buffer only (B lanes). Bound antibodies were detected with 125I-labeled sheep anti–mouse antibodies.
Mentions: Preliminary ELISA tests had shown that antibody EH5 was able to discriminate between E. histolytica and E. dispar (data not shown). Fig. 1 shows the immunoblot results obtained with membrane preparations from different xenically and axenically grown E. histolytica and E. dispar strains using antibody EH5. In addition, a membrane preparation from Trichomonas vaginalis, another protist parasite which is only distantly related to E. histolytica and E. dispar, was prepared and included. The immunoblots show strong reactivity for all E. histolytica strains of antigens migrating as a polydisperse band, whereas E. dispar bound to antibody EH5 weakly, and T. vaginalis hardly at all. In addition, the signals were stronger when the amebas had been grown axenically.

Bottom Line: Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A.Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess.Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

View Article: PubMed Central - PubMed

Affiliation: Institute for Specific Prophylaxis and Tropical Medicine, A-1095 Vienna, Austria.

ABSTRACT
A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

Show MeSH
Related in: MedlinePlus