RESUMO
The reactivity of sera from experimentally infected animal was studied from 5-60 days postinoculation to determine which of the E. histolytica antigens are recognized frequently to infection. Crude extract of E. histolytica trophozoites was used and sera were examined by immunoelectrotransference assay. It was observed that sera recognized polypeptide with 70 kDa molecular mass after 15 days postinoculation onward and later 14 to 24 polypeptide with molecular weight of 110-22 kDa were recognized. All the amebic antigens (polypeptides) could be recognized by sera till 60 days postinoculated animals. Significance of expression of different amebic polypeptides in terms of pathogenesis needs further investigations.
Assuntos
Animais , Reações Antígeno-Anticorpo , Antígenos de Protozoários/química , Entamoeba histolytica/imunologia , Entamebíase/imunologia , Enterite/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C3H , Peso MolecularRESUMO
A mouse monoclonal antibody, Eh208C2-2 MAb, raised against whole cell antigens of Entamoeba histolytica trophozoites of the pathogenic strain HM-1: IMSS and polyclonal antisera (PAb) against membrane antigens of E. histolytica trophozoites of strain HTH-56: MUTM were screened against a cDNA library of the pathogenic strain, SFL3. The monoconal antibody detected many phage plaques expressing an E. histolytica protein. The DNA sequence encoding the protein was approximately 55% identical, over 1,100bp, to Trichomonas vaginalis pyruvate: ferredoxin oxidoreductase (PFOR) and pyruvate: flavodoxin oxidoreductase from Klebsiella pneumoniae, Anabaena variabilis and Enterobacter agglomerans. Two of seven clones detected by mouse polyclonal antisera also encoded this protein. Two others encoded Entamoeba Hsp70, another encoded Entamoeba alkyl-hydroperoxide reductase and the remaining two were unidentified sequences. Entamoeba PFOR is an abundant, antigenic protein which may be a useful target for the development of protective host immune responses against invasive amebiasis.
Assuntos
Sequência de Aminoácidos/genética , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Protozoários/genética , Sequência de Bases , DNA Complementar/genética , Entamoeba histolytica/genética , Entamebíase/imunologia , Biblioteca Gênica , Cetona Oxirredutases/genética , Camundongos , Dados de Sequência Molecular , Piruvato SintaseRESUMO
The HK-9 strain of Entamoeba histolytica was cultured axenically. A crude extract of the trophozoites was used as antigen in the enzyme-linked immunosorbent assay (ELISA) for the determination of antiamoebic IgG, IgM, IgA and IgG subclasses in sera of patients with E. histolytica infection. Sera from amoebiasis patients had significantly higher mean ELISA values for all classes and subclasses of immunoglobulins examined compared with healthy controls. The ELISA for IgG, IgM and IgA was positive for 76 (97.4%), 34 (43.6%) and 62 (79.5%) respectively of 78 amoebiasis sera. Analysis of IgG subclasses by ELISA showed that 76.7%, 25%, 33.3% and 66.6% of the patients were positive for antiamoebic IgG1, IgG2, IgG3 and IgG4 respectively. IgG1 and IgG4 were the dominant IgG subclasses involved in amoebiasis. The role of antiamoebic antibodies in amoebiasis is discussed.
Assuntos
Amebíase/imunologia , Anticorpos Antiprotozoários/análise , Células Cultivadas , Entamebíase/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina A/análise , Imunoglobulina G/análise , Imunoglobulina M/análise , Abscesso Hepático Amebiano/imunologiaRESUMO
Two methods used for the purification of specific antigen for amoebiasis were affinity chromatography and agarose gel electrophoresis. The IgG fraction of the serum from a patient with amoebic liver abscess showing a specific band in the IEP test was coupled to CNBr-activated Sepharose 4B. After passage through the immunoadsorbent column of the crude or partially purified extract of Entamoeba histolytica, followed by acid dissociation, specific antigen was obtained. This antigen gave only one band in the IEP test against the serum which gave multiple bands upon testing with the crude antigen. With the agarose gel electrophoresis, fraction 2 from the slit was found to contain specific antigen. Rabbits immunized with the excised agarose strip of this fraction produced antibodies giving 2 precipitating bands in the IEP test, one of which was specific for amoebiasis.
Assuntos
Amebíase/imunologia , Animais , Antígenos/isolamento & purificação , Entamoeba histolytica/imunologia , Entamebíase/imunologia , Epitopos , Humanos , Imunoeletroforese , CoelhosRESUMO
The indirect hemagglutination test (IHA) and counterimmuno-electrophoresis (CIE) were used to test for antibodies to Entamoeba histolytica in human sera using antigens from axenic cultures of the HK9 strain. Correlation between the test was excellent with most sera from patients with amoebiasis demonstrating precipitin lines by CIE at IHA titers considered diagnostically positive, larger than or equal to 1:128. Precipitin reactions with positive sera were also compared by CIE, immunoelectrophoresis (IEP) and two-dimensional electrophoresis is (2D-IEP). The greatest number of antigen-antibody components were demonstrated by 2D-IEP. Further studies utilizing 2D-IEP should be of value in the antigenic analysis of E. histolytica.