== Three adult (6-week-old) female BALB/c mice were bled and immunized intraperitoneally four times at 2-week intervals with 4 107spores per 100 l emulsified at a 1:1 ratio with Freund’s total adjuvant (Calbiochem, La Jolla, CA) for the first inoculation and with Freund’s incomplete adjuvant (Sigma, St

== Three adult (6-week-old) female BALB/c mice were bled and immunized intraperitoneally four times at 2-week intervals with 4 107spores per 100 l emulsified at a 1:1 ratio with Freund’s total adjuvant (Calbiochem, La Jolla, CA) for the first inoculation and with Freund’s incomplete adjuvant (Sigma, St. varieties, including both immunologically normal and simian immunodeficiency computer virus (SIV)-infected macaques (Macaca mulatta,Macaca cyclopies, andMacaca nemestrina) (17,23,24,32).E. bieneusiis 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 computer virus (HIV)/AIDS (11,25,28,29) and macaques with SIV/AIDS (6,7). We have previously demonstrated thatE. bieneusistrains isolated from macaques and humans are morphologically, genetically, and antigenically indistinguishable (7,24). Human- and rhesus-derivedE. bieneusisequences 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 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that spores from these two mammal-infecting species possess different specific densities and different karyotypes (unpublished data).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 propagateE. bieneusiin 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 humanE. bieneusihave been reported (2), but they are unavailable commercially. To our knowledge, the production of MAbs againstE. bieneusiisolates of rhesus macaque source has not been 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 (Macaca mulatta) were housed at the New England Regional Primate Study Center in accordance with the standards of the American Association of Laboratory Animal Care and Harvard Medical School’s Animal Care and Use Committee. Fecal samples were collected daily and were analyzed forE. bieneusishedding 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 ofE. bieneusispores. (i) Salt-Percoll-sucrose centrifugation. == Fecal specimens were processed as explained previously (33), with the following modifications. Briefly, feces were Zoledronic acid monohydrate 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 gfor 40 min and washed Rabbit polyclonal to MICALL2 four occasions with distilled water (3,200 g, 20 min). The pellet was mixed with PBS and saturated sodium chloride (the final concentration of sodium chloride was 75%) and centrifuged at 1,000 gfor 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 coating was collected, its sodium chloride concentration was modified to 50%, and the spores were pelleted at 3,200 gfor 30 min. The pellet was washed one more time (3,200 g, 20 min) and then resuspended in PBS. For Percoll centrifugation, the samples were mixed with 72% isotonic Percoll (3 ml spores plus 30 ml 72% Percoll) and centrifuged for 60 min at 14,000 g. The coating above the sediments (B, 2 to 2.5 ml) and the middle coating (M, 26 ml) were collected separately and washed one more time (3,200 g, 20 min). Both layers were loaded on a 30 to 60% (wt/wt) sucrose gradient and centrifuged for 24 h at 77,000 g. Spores were collected having a 20-gauge needle and pelleted at 77,000 gfor 60 min. Spores were washed twice with PBS (18,000 g, 3 Zoledronic acid monohydrate min). == (ii) Preformed iodixanol gradient centrifugation. == OptiPrep Zoledronic acid monohydrate denseness gradient medium (60% [wt/vol] answer Zoledronic acid monohydrate of iodixanol in water) was purchased from Sigma (St. Louis,.