Development of a diagnostic test for Entamoeba histolytica using idiotype expression in human.

The protozoan parasite Entamoeba histolytica is the etiological agent of human amebiasis. The pathology of the disease starts with the cytolysis of the host target cells by amoebae. It is initiated by the adhesion of trophozoites to the host cells, through surface lectin via specific receptors. These adherence lectins have been demonstrated to be highly conserved, and can be recognised by serum antibodies from patients with invasive amebiasis. Some of these molecules have been used as antigens in serologic studies, which has been very helpful in the diagnosis of invasive intestinal amebiasis. However, false-positive serologic reactivity can occur using E. histolytica extracts and purified antigens. Additional problems are because the extracts display a great enzymatic activity. Several diagnostic methods, using different molecules and techniques, have been described. However, the problem still remains since these tests are not capable of differentiating between amoebic liver abscess (ALA) and intestinal amebiasis.Here, the research has been addressed to the 66-kDa antigen, which is a part of the outer membrane proteins from the E. histolytica strain HM1-IMSS trophozoites. First of all, we characterized the 66-kDa antigen in order to prove the relevance. We found that the 66-kDa antigen is a part of the plasma membranes and is distributed rather homogeneously on the cell surface of trophozoites. Apparently, the 66-kDa antigen is a glycoprotein. Using a monoclonal antibody (MAb), we found 25% of inhibition in the erythrophagocytosis by the trophozoites. Starting form one monoclonal antibody, we prepared an anti-idiotype (anti-Id) antibody reagent, with the purpose of searching for the different expressions of the idiotype between the sera from ALA and the intestinal amebiasis patients. Moreover, we produced the antibody Ab3 that is capable of recognising the 66-kDa antigen; it means that the Ab2 displays the internal image of the antigen. We found that 91.6% of the serum from ALA patients displayed the expression of the Id. In contrast, 15.7% of the E. histolytica asymtomatic cyst carriers displayed the Id expression, 6.6% of the patients with another parasite infection, and 11% of the negative controls (serum from umbilical cords of newborn babies). Our results showed that the expression of the Id could be differentiated among the AHA patients from the other groups with a 91.6% sensibility and 88.3% specificity.

Identification of a 35 kDa glycoprotein from Entamoeba histolytica by sera from patients with amoebic liver abscess and with mouse monoclonal antibody.

In this work, we explored the relevance of a 35 kDa glycoprotein (Gm) of the outer membrane from E. histolytica in the diagnosis of the amoebic liver abscess (ALA) through ELISA and immunoblotting. We were interested in defining the relevance of this antigen in the immune response in patients with amoebic liver abscess and in exploring whether the mouse monoclonal antibody against this 35 kDa glycoprotein recognises the same epitope. We found that 87% of ALA patients had raised antibody levels to Gm antigen, whereas none of the healthy control subjects presented this same increase. We also found 90% sensitivity, 100% specificity, 100% positive predictive value, 90% negative predictive value, and 90% prevalence value for this Gm antigen. Nonetheless, we did not find any statistically significant differences in the levels of immunoglobulins against Gm, although IgG showed a tendency to increase, probably because we are dealing with a secondary immune response. Using electroimmunotransfer blot assay, we found that sera from ALA patients recognise the 35 kDa Gm protein in the same way as it is recognised by the mouse monoclonal antibody, suggesting that is a relevant molecule for the diagnosis of amebiasis, and eventually could lead to its use as protection against the disease.