These results were consistent with nested PCR results

These results were consistent with nested PCR results. This is the first report of the development of MAbs against derived from rhesus macaques which cross-react with strains that infect humans. individuals with AIDS (4, VEGFA 10, 39). has also been recognized in immunologically healthy individuals with diarrhea (3, 12, 19, 21, 31, 35) and in individuals receiving immunosuppressive therapy (15, 18, 26, 30, 34). has also been described as infecting additional mammalian varieties, including both immunologically normal and simian immunodeficiency disease (SIV)-infected macaques (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 being immunodeficiency disease (HIV)/AIDS (11, 25, 28, 29) and macaques with SIV/AIDS (6, 7). We have previously demonstrated that strains isolated from macaques and humans are morphologically, genetically, and antigenically indistinguishable (7, 24). Human being- and rhesus-derived sequences share 99.5% nucleic acid sequence identity over Oleuropein a 2.0-kb fragment of the ribosomal gene complex (5). However, recent data from our laboratory shown that spores from these two mammal-infecting species possess different specific densities and different karyotypes (unpublished data). In the absence of the ability to propagate 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 additional laboratories (1, 8, 20) as well as by our group (33). Two monoclonal antibodies (MAbs) against human being have been reported (2), but they are unavailable commercially. To our knowledge, the production of MAbs against isolates of rhesus macaque source has not been Oleuropein reported. With this communication, we describe the concentration and purification of spores from feces of macaques in adequate quantities to generate several well-characterized specific MAbs. MATERIALS AND METHODS Fecal samples. All rhesus macaques (dropping by nested PCR relating to a previously explained process (5, 24, 40, 41). For purification and MAb production, feces from SIV-infected rhesus macaques were collected in phosphate-buffered saline (PBS) and stored at 4C for further control. Purification of spores. (i) Salt-Percoll-sucrose centrifugation. Fecal specimens were processed as explained previously (33), with the following modifications. Briefly, feces were homogenized in 0.01 M PBS, pH 7.2 to 7.4 (1:5 to 1 1:10), and serially filtered through American standard sieves (pore sizes, 425, 180, 100, and 63 m; Newark Wire Fabric Organization, Newark, NJ). The spores were pelleted at 3,200 for 40 min and washed four instances with distilled water (3,200 for 15 min. In order to increase the recovery of spores, the pellet was processed again with a final sodium chloride concentration of 85%. The middle layer was collected, its sodium chloride Oleuropein concentration was modified to 50%, and the spores were pelleted at 3,200 for 30 min. The pellet was washed one more time (3,200 for 60 min. Spores were washed twice with PBS (18,000 (16, 37). The spores were collected and resuspended in PBS. The recovery of spores at each step was monitored by an immunofluorescence assay (IFA) with rabbit polyclonal antibodies against human as explained previously (33). TEM. Purified spores were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 18 to 24 h. The samples were rinsed in buffer and postfixed in 1% osmium tetroxide made up of 0.8% potassium ferricyanide for 1 h. Samples were completely dehydrated in a graded series of ethanol. The spores were infiltrated with epoxy plastics according to the Mollenhauer formulation (27) and then cured at 60C for 48 h. The blocks were sectioned on a Leica Ultracut R microtome, and the grids were stained with saturated uranyl acetate and lead Oleuropein citrate. Grids were viewed and photographed on a Phillips CM-10 electron microscope. The purity of the spores was examined under a low magnification by transmission electron microscopy (TEM). The purities of different bands were calculated by counting spores, bacteria, and other debris on each section. Production of monoclonal antibodies. Three adult (6-week-old) female BALB/c mice were bled and immunized intraperitoneally four occasions at 2-week intervals with 4 107 spores per 100 l emulsified at a.

Comments are closed.