Characterization of monoclonal antibody specific to entamoeba histolytica cells and erythrophagosomes

Abstract

Objective: Entamoeba histolytica is a pathogenic protozoan which causes the third most deaths from parasitic diseases worldwide. Monoclonal antibodies (mAbs), which are specific to E. histotytica were successfully produced previously. In this study, E. histolytica specific mAbs were characterized for localization of mAb-binding partner in the live E. histolytica trophozoites. Methods: Trophozoite cells were stained with a mAb and analyzed by fluorescent microscopy and flow cytometry, to investigate the localization of a mAb reacting protein in the pathogen cells. mAbs were further tested with proteins extracted from cellular fractions of E. histolytica using ELISA. Trophozoites were optimally challenged with red blood cells and proteins were solubilized from the erythrophagocytosed cells by detergents. Soluble proteins were incubated with protein G-beads linked mAb#32 or a control mAb#19. E. histolytica proteins pulled down by mAbs were analyzed with SDS-PAGE and visualized by silver stain. Results and Discussion: The results showed that most of the mAbs bind to cytoplasmic proteins, with 4 clones interacting with the membrane proteins and two clones reacting with phagosomes located inside the cells. It was found that challenging E. histolytica cells with 0.25% human red blood cells for 1 hour at 37°C resulted in the highest yield of the erythrophagosomes. SDS-PAGE revealed E. histolytica proteins with a molecular masses of about 60-65 kDa and more than 170 kDa, which are successfully pulled down by mAb #32. These proteins will be subjected to further protein identification by mass spectrometry and may play role erythrophagocytosis, which is a virulent factor of the parasite during tissue invasion.