Production and Characterization of Monoclonal Antibodies Against a Highly Immunogenic Fraction of Entamoeba histolytica (NIH:200) and Their Application in the Detection of Current Amoebic Infection

Abstract

Six monoclonal antibodies (MAbs) were produced against a highly immunogenic fraction derived by the chromatographic separation of the soluble preparation of axenic Entamoeba histolytica (strain NIH:200) trophozoites. Isotype characterization of the six MAbs revealed that four belonged to the IgM class and one each to the IgG1 and the IgG2a subclasses. The immunoreactivity patterns and the specificity of the MAbs with homologous and heterologous antigens were analyzed by the enzyme-linked immunotransfer blot technique and by the enzyme-linked immunosorbent assay. The MAbs reacted intensely with isolates of E. histolytica (strain NIH:200 as well as a local isolate MX1) but showed no reactivity with Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Entamoeba hartmanni, free-living amoeba (Acanthamoeba harticolus) and other enteric parasites. Using the IgG1 MAb as a detecting antibody, a polyclonal-monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of E. histolytica antigens in stool samples of infected patients. The detection limit of the assay was 8 ng of amoebic antigen. This test was found to be specific and sensitive and yielded 100% positive results in cases with amoebiasis but did not react with controls included in the evaluation. The MAb-based enzyme-linked immunosorbent assay developed in this study will be an important test for the diagnosis of E. histolytica in the feces of infected humans; however, the limitation of the test is the inability to discriminate the pathogenic status of the amoeba detected in the stool.

The Investigation of Entamoeba histolytica Prevalence in Some Villages of Sivas by ELISA Method

“…Some records are available that show the occurrence of these organisms in our country, but no data has been found in this region (4,(22)(23)(24)26). No cross reaction was detected with other luminal protozoa (E. histolytica/dispar, E. coli, B. hominis, G. intestinalis, I. butschlii) as previously reported (25). Our study indicates that E.dispar may be more common in our region than in other countries (26).…”

The 29-Kilodalton Thiol-Dependent Peroxidase of Entamoeba histolytica Is a Factor Involved in Pathogenesis and Survival of the Parasite during Oxidative Stress

“…Electrophoretically, 80-g aliquots of proteins from different transfected trophozoite lysates were resolved in a 10% polyacrylamide gel under reducing conditions and transferred onto a nitrocellulose membrane (BioRad). The membrane was washed with Tris-buffered saline, pH 7.2, blocked with 3% bovine serum albumin (Sigma) at 37°C for 2 h, and reacted with a 1:1,000 dilution of NICED 11 ascites (monoclonal antibody specific for the 29-kDa protein of E. histolytica) (33). Fibronectin (1,34) antibody was used to check the equal loading of proteins.…”

Antigens of diagnostic and prophylactic importance inEntamoeba histolytica

“…The rationale for such an approach was due to the principle that (i) Mabs generated against specific epitopes, will help in development of a diagnostic test for amoebiasis cases directly from stool samples, (ii) to analyse all the Mabs for their specific role in the differentiation of pathogenic E. histolytica from non pathogenic E. dispar directly from the clinical specimens and (iii) to purify the antigens to the well characterized Mabs and assess their importance in immunoprophylaxis studies. Of the six, one Mab (NICED 11) which showed IgG1 isotype, high agglutination activity and reaction with single epitope present at 29 kDa region of FI antigen was used in multi layer ELISA for the detection of amoebic antigen in stool specimen (4). The test could detect as low as 8 ng of amoebic antigen in faecal samples (equal to two trophozoites) and was found to be much more sensitive and specific than the previously described conventional methods.…”