Production and Characterization of Monoclonal Antibodies against Enterocytozoon bieneusi Purified from Rhesus Macaques

Abstract

Enterocytozoon bieneusi spores derived from rhesus macaque feces were purified by serial salt-Percollsucrose-iodixanol centrifugation, resulting in two bands with different specific densities of 95.6% and 99.5% purity and with a recovery efficiency of 10.8%. An ultrastructural examination revealed typical E. bieneusi spores. Twenty-six stable hybridomas were derived from BALB/c mice immunized with spores and were cloned twice by limiting dilution or growth on semisolid medium. Four monoclonal antibodies (MAbs), reacting exclusively with spores, were further characterized. These MAbs specifically reacted with spores present in stools of humans and macaques, as visualized by immunofluorescence, and with spore walls, as visualized by immunoelectron microscopy. A blocking enzyme-linked immunosorbent assay and Western blotting revealed that the epitope recognized by 8E2 was different from those recognized by 7G2, 7H2, and 12G8, which identified the same 40-kDa protein. These MAbs will be valuable tools for diagnostics, for epidemiological investigations, for host-pathogen interaction studies, and for comparative genomics and proteomics.Enterocytozoon bieneusi is clinically the most significant microsporidian parasite associated with persistent diarrhea and wasting in individuals with AIDS (4, 10, 39). E. bieneusi has also been identified in immunologically healthy patients with diarrhea (3,12,19,21,31,35) and in individuals receiving immunosuppressive therapy (15,18,26,30,34). E. bieneusi has also been described as infecting other mammalian species, including both immunologically normal and simian immunodeficiency virus (SIV)-infected macaques (Macaca mulatta, Macaca cyclopies, and Macaca nemestrina) (17,23,24,32). E. bieneusi is found within the cytoplasm of epithelial cells of the gallbladder, bile ducts, and the small intestine, causing a proliferative cholecystitis, serositis, cholangiohepatitis, and enteropathy, respectively, in humans with human immunodeficiency virus (HIV)/AIDS (11,25,28,29) and macaques with SIV/ AIDS (6, 7). We have previously shown that E. bieneusi strains isolated from macaques and humans are morphologically, genetically, and antigenically indistinguishable (7, 24). Humanand rhesus-derived E. bieneusi sequences share 99.5% nucleic acid sequence identity over a 2.0-kb fragment of the ribosomal gene complex (5). However, recent data from our laboratory demonstrated that spores from these two mammal-infecting species have different specific densities and different karyotypes (unpublished data).In the absence of the ability to propagate E. bieneusi in vitro or in vivo (38), feces from infected humans or rhesus macaques are the only available source of spores. Purification has not been easy because of the size of the spores. Several methods to purify spores from feces have been described by other laboratories (1,8,20) as well as by our group (33). Two monoclonal antibodies (MAbs) against human E. bieneusi have been reported (2), but they are unavailable commercially. To our knowledge, the produ...

Antibody-Enhanced, Fc Gamma Receptor-Mediated Endocytosis of Clostridium difficile Toxin A

“…Murine hybridomas secreting anti-TcdA antibodies were generated using nTcdA as an immunogen as described elsewhere, with modifications (68). The hybridoma supernatants were screened for antigen-binding capacity by performing an enzyme-linked immunosorbent assay (ELISA) using microplates coated with 0.5 g/ml of rTcdA.…”

Sensitivity and Specificity of a Monoclonal Antibody-Based Fluorescence Assay for Detecting Enterocytozoon bieneusi Spores in Feces of Simian Immunodeficiency Virus-Infected Macaques

“…All three E. bieneusi-specific MAbs reacted with the simian E. bieneusi spore wall but not with other microsporidia, as shown elsewhere (21,28). The MAb of IgM had a titer of 1:4, whereas the two IgG antibodies had titers of Ͼ1/1,000, as determined by indirect IFA on feces of SIV-infected macaques containing E. bieneusi spores.…”

“…Rabbit polyclonal E. bieneusi antibody was used as a positive control (20). 1B7 and 2G4 MAbs were produced against human E. bieneusi spores, and 12G8 MAb was produced against monkey E. bieneusi spores, as described elsewhere (21,28). To determine the dilution of the MAbs used, the culture supernatants were titrated on feces positive for E. bieneusi spores (data not shown).…”

Serial Propagation of the Microsporidian Enterocytozoon bieneusi of Human Origin in Immunocompromised Rodents

“…The spores were enriched by a salt flotation step, followed by a continuous sucrose gradient centrifugation step, which separated the spores from the fecal material, bacteria, and yeasts, as previously described. The purified spores were detected by an indirect fluorescentantibody (IFA) assay using antibodies specific for E. bieneusi (16,25) and were further characterized as E. bieneusi by PCR (21). The purity of each spore preparation was also evaluated by microbial culture and by transmission electron microscopy, as previously described (16).…”

“…The inability to serially propagate E. bieneusi in cultured cells and/or in animal models has limited laboratory investigations and hindered research progress on this pathogen and the search for an effective therapy. Recent progress, including the development of purification and concentration procedures for spores from stools of infected humans (11,16), has led to the production of immune reagents (2,25) and to renewed attempts to propagate E. bieneusi in cell culture and in small laboratory animals. We report here the successful transmission and serial propagation of E. bieneusi obtained from humans in several rodent models.…”