Studies on the Antigens of Entamoeba histolytica Schaudinn, 1903

Strachan, William David (1990) Studies on the Antigens of Entamoeba histolytica Schaudinn, 1903. PhD thesis, University of Glasgow.

Abstract

Twenty one monoclonal antibody-secreting hybridoma cell lines were produced from fusions between mouse myeloma cells and spleen cells from mice immunised with axenically-cultivated Entamoeba histolytica. These monoclonal antibodies (Mabs) were used in the subsequent study of the antigens for which they were specific. The subcellular locations of these antigens within E. histolytica trophozoites were visualised by use of the Mabs in indirect fluorescent antibody tests (IFAT). Three of the Mabs recognised antigens located on the surface of the amoebae; confirmed by positive results in IFAT experiments using live E. histolytica. These Mabs recognised only a percentage of cells (the percentage was different for each Mab) present in cultures. This heterogeneity of antigen expression within a culture was not a stable property of the cells, since cloned lines were also heterogeneous when tested. The phenomenon, which occurred in all isolates recognised by these Mabs, may be related to the cell cycle or, more likely, be due to subpopulations of cells arising in the cultures. Two of the Mabs were specific for antigens related to the vacuolar system of the amoeba while the remainder recognised other internal components of the amoeba. The Mabs were tested for their ability to recognise antigens in IFAT tests using other protozoan cell types, including other members of the genus Entamoeba. Two of the Mabs recognised all cell types tested, including a mammalian cultured cell line. Eight Mabs failed to cross-react with any of the cells tested, while a further nine reacted with either Entamoeba invadens (a reptile pathogen) or Entamoeba moshkovskii (free-living in sewage) or both. These experiments demonstrated a high degree of antigenic relatedness between some members of the quadrinucleate cyst group of the genus Entamoeba; E. histolytica, E. invadens and E. moshkovskii, but not E. hartmanni. The identical pattern of Mab cross-reaction obtained with Entamoeba coli (from man) and Entamoeba muris (from mice) reflected the morphological indistinguishability of these two members of the octonucleate cyst group of Entamoebae. Fifteen isolates of E. histolytica, other than strain 200:NIH (against which these Mabs were raised), were tested in IFAT experiments using the Mabs. In general, very little antigenic heterogeneity was observed. The failure of the three surface-staining Mabs to recognise some strains may have been due to an undetectably low number of positive cells. Apart from these, only three other Mabs failed to recognise all isolates. In subsequent IFAT experiments involving a larger sample of E. histolytica isolates, two of these Mabs, 22.3 and 22.5, were shown to react specifically with amoebae belonging to pathogenic zymodemes. The nature of the pathogen-specific antigens recognised by these Mabs, is not known. Approximate molecular weights of some of the antigens recognised by these Mabs, were determined using the Mabs as probes in electro-immunotransfer blotting of amoebic lysates previously subjected to polyacrylamide gel electrophoresis. Values were obtained using thirteen of the Mabs and ranged from 12 kD to over 300 kD with the majority between 95 and 130 kD. Mabs found not to cross-react with other tested human protozoan parasites (selected on the basis of previous IFAT experiments) were tested for their ability to detect E. histolytica antigen as primary capture reagents in a double sandwich enzyme-linked immunosorbent assay (ELISA). Two Mabs, 24.7 and 29.3, consistently detected antigen (amoebic lysate) down to a concentration of 0.1 mug total protein per ml of sample, indicating their potential usefulness in an immunodiagnostic test for amoebiasis.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Cellular biology, Immunology
• Date of Award: 1990
• Depositing User: Enlighten Team
• Unique ID: glathesis:1990-78046
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 30 Jan 2020 15:42
• Last Modified: 30 Jan 2020 15:42
• URI: https://theses.gla.ac.uk/id/eprint/78046